阅读说明：本技术 硝基呋喃类化合物、药物组合物及其制备方法和用途 (Nitrofuran compound, pharmaceutical composition, preparation method and application thereof ) 是由 王元相 张小雷 李骅轩 欧阳淑敏 于 2021-04-15 设计创作，主要内容包括：本发明公开了硝基呋喃类化合物、药物组合物及其制备方法和用途。该硝基呋喃类化合物具有如式(Ⅰ)所示结构：本发明提供的硝基呋喃类化合物具有类药性优势骨架,具备成药性等特征,对STAT3蛋白的选择性高,对STAT3有显著的抑制作用,可作为STAT3特异性抑制剂；另外,该硝基呋喃类化合物能够显著减小体内肿瘤的体积和重量,明显抑制肿瘤的生长增殖；除此之外,该硝基呋喃类化合物或其药学上可以接受的盐或溶剂合物与EGFR抑制剂联合用药时,可以延缓EGFR药物获得性耐药的产生、延长其临床使用寿命,具有重要的临床意义。(The invention disclosesNitrofurans compounds, pharmaceutical compositions, methods of preparation and uses thereof are disclosed. The nitrofuran compound has a structure shown as a formula (I): the nitrofuran compound provided by the invention has a skeleton with the advantage of drug property, has the characteristics of drug forming property and the like, has high selectivity on STAT3 protein, has a remarkable inhibiting effect on STAT3, and can be used as a STAT3 specific inhibitor; in addition, the nitrofuran compound can obviously reduce the volume and weight of tumors in vivo and obviously inhibit the growth and proliferation of the tumors; in addition, when the nitrofuran compound or the pharmaceutically acceptable salt or solvate thereof and the EGFR inhibitor are used together, the generation of acquired drug resistance of the EGFR drug can be delayed, the clinical service life of the EGFR drug can be prolonged, and the nitrofuran compound or the pharmaceutically acceptable salt or solvate thereof has important clinical significance.)

1. The nitrofuran compound is characterized by having a structure shown as a formula (I):

wherein:

R₁、R₂、R₃、R₄independently selected from hydrogen, halogen, cyano, nitro, amino, hydroxy, trifluoromethyl, substituted or unsubstituted C_1-6Alkyl, substituted or unsubstituted C_1-6Alkoxy, substituted or unsubstituted C_1-6Alkylamino, substituted or unsubstituted C_3-8Cycloalkyl, substituted or unsubstituted C_3-8Cycloalkoxy, substituted or unsubstituted C_3-8Cycloalkylamino, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted 3-to 8-membered heterocyclyl with 1 to 4 heteroatoms selected from N and O, -COR^a、-CO₂R^a、-CONR^aR^b、-NR^aC(O)R^b、-NR^aSO₂R^b、-SR^a、-SOR^a、-SO₂R^a、-SO₂NR^aR^b、-OC(O)R^aor-OC (O) NR^aR^b，

R^aAnd R^bIndependently of one another is hydrogen, C_1-6Alkyl radical, C_3-6Cycloalkyl, aryl or heteroaryl;

R₅is hydrogen, halogen, cyano, nitro, amino, hydroxy, trifluoromethyl, substituted or unsubstituted C_1-8Alkyl, substituted or unsubstituted C_1-8Alkoxy, substituted or unsubstituted C_1-8Alkylamino, substituted or unsubstituted C_3-8Cycloalkyl, substituted or substituted C_3-8Cycloalkoxy, substituted or unsubstituted C_1-8Alkylamino, substituted or unsubstituted aryl or heteroaryl;

R₆、R₇、R₈、R₉independently selected from hydrogen, halogen, cyano, nitroAmino, hydroxy, trifluoromethyl, C_1-3Alkoxy radical, C_1-3An alkylamino group.

2. The nitrofurans of claim 1, wherein R is₁、R₂、R₃、R₄Is substituted at least at 1 position with the following substituents: halogen, cyano, amino, nitro, hydroxy, trifluoromethyl, C_1-3Alkyl radical, C_1-3Alkoxy radical, C_1-3Alkylamino, -COR^a、-CO₂R^a、-CONR^aR^b、-NR^aC(O)R^b、-NR^aSO₂R^b、-SR^a、-SOR^a、-SO₂R^a、-SO₂NR^aR^b、-OC(O)R^a、-OC(O)NR^aR^b。

3. Nitrofurans according to claim 1 or 2, characterized in that R₁、R₂、R₃、R₄Independently selected from H, halogen, cyano, nitro, amino, hydroxy, trifluoromethyl, C_1-6Alkyl radical, C_1-6Heteroalkylalkoxy, C_1-6Alkylamino radical, C_3-8Cycloalkyl radical, C_3-8Cycloalkoxy, C_3-8Cycloalkylamino, aryl, heteroaryl, 3-to 8-membered heterocyclyl containing 1-2 heteroatoms selected from N and O.

4. The nitrofurans of claim 1, wherein R is₅Is substituted at least at 1 position with the following substituents: halogen, cyano, amino, nitro, hydroxy, trifluoromethyl, methylthio, C_1-3Alkyl radical, C_1-3Alkoxy or C_1-3An alkylamino group.

5. The nitrofurans of claim 1, wherein R is₁、R₂、R₃、R₄、R₅The aryl or hetero group in (1)Aryl is independently selected from the group consisting of: furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, benzofuryl, benzothienyl, benzoxazolyl, benzothiazolyl, phenyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, quinolinyl, or naphthyl.

6. A method for preparing the nitrofurans compound according to any one of claims 1 to 5, comprising the steps of:

s1: dissolving the formula (1) and the formula (2) in a solvent, and generating an intermediate (3) after reaction;

s2: inserting a side chain into the intermediate (3) to generate an intermediate (4);

s3: reducing the intermediate (4) to generate an intermediate (5), and then carrying out condensation reaction to obtain the nitrofuran compound;

7. use of the nitrofurans compound or the pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 5 in preparation of a medicament for inhibiting STAT3 protein activity.

8. The use according to claim 6, wherein the nitrofurans or pharmaceutically acceptable salts or solvates thereof is used for preparing a medicament for treating abnormal cell proliferation, morphological change and hyperkinesia of STAT3 overexpression or treating angiogenesis or cancer metastasis;

the application of the nitrofurans or the pharmaceutically acceptable salt or solvate thereof in preparing the medicines for inhibiting the tyrosine phosphorylation level of STAT 3.

9. Use of the nitrofurans of claims 1 to 5 or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicament for inhibiting tumor growth and proliferation.

10. A pharmaceutical composition comprising the nitrofurans of claims 1 to 5 or a pharmaceutically acceptable salt or solvate thereof, and an EGFR inhibitor.

Technical Field

The invention relates to the field of pharmaceutical chemistry and pharmacotherapeutics, in particular to a nitrofuran compound, a pharmaceutical composition, a preparation method and application thereof.

Background

The transcription factor STAT3 is a bifunctional protein, has dual functions of signal transduction and transcriptional activation, and is responsible for regulating and controlling a series of important physiological processes of cell growth, proliferation, differentiation, apoptosis and the like. However, studies find that STAT3 has persistent activation and abnormally high expression, can induce cell proliferation, invasion and migration, inhibit apoptosis, promote angiogenesis, and play an important role in the process of tumor generation, development and metastasis. Therefore, the development of targeted STAT3 inhibitors has been a hot spot in the frontier of antitumor drug research.

Currently, several targeted STAT3 drug candidates have entered phase I/II clinical trials, such as: s31-201, STA-21, Niclosamide, C188-9, OPB-31121, BP-1-102 and pyrimethanamine, but no drug is yet approved for clinical treatment. Where most compounds under investigation were non-specific STAT3 inhibitors, lacking selectivity; on the other hand, the proliferation inhibition effect of most compounds on STAT3 high-expression tumor cell lines is still at micromolar level; moreover, many compounds also face the problem of drug forming properties such as low oral bioavailability (e.g. Niclosamide), poor solubility (e.g. C188-9) and unstable structure (e.g. BP-1-102), and further structural optimization is still needed to improve drug forming properties.

In conclusion, specific inhibitors targeting STAT3 have been the focus of research and development (e.g., European Journal of Medicinal Chemistry,2020,187,111922), but the drugs developed so far all have the disadvantages of small drug effect, low selectivity, poor drug potency, and the like, in different degrees and different aspects, and limit the clinical application and later development of STAT3 inhibitors. While STAT3 is a promising tumor treatment target, the development of a new class of compounds which can specifically inhibit STAT3 and have the characteristics of high selectivity, strong drug effect, good drug property and the like is urgently needed in the field.

Disclosure of Invention

The invention aims to overcome the defects or shortcomings of small efficacy, low selectivity, poor drug property and the like of the existing STAT3 and provides a nitrofuran compound. The nitrofuran compound provided by the invention has a skeleton with the advantage of drug property, has the characteristics of drug forming property and the like, has high selectivity on STAT3 protein, has a remarkable inhibition effect on STAT3, and can be used as a STAT3 specific inhibitor; in addition, the nitrofuran compound can obviously reduce the volume and weight of tumors in vivo and obviously inhibit the growth and proliferation of the tumors; in addition, when the nitrofuran compound or the pharmaceutically acceptable salt or solvate thereof and the EGFR inhibitor are used in combination, the generation of acquired drug resistance of the EGFR drug can be delayed, the clinical service life of the EGFR drug can be prolonged, and the nitrofuran compound or the pharmaceutically acceptable salt or solvate thereof has important clinical significance.

The invention also aims to provide a preparation method of the nitrofuran compound.

The invention also aims to provide application of the nitrofurans or pharmaceutically acceptable salts or solvates thereof in preparing a medicament for inhibiting STAT3 protein activity.

The invention also aims to provide the application of the nitrofurans compound or the pharmaceutically acceptable salt or solvate thereof in preparing the medicines for inhibiting the growth and proliferation of tumors

It is another object of the present invention to provide pharmaceutical compositions.

In order to achieve the above purpose of the present invention, the present invention provides the following technical solutions:

the nitrofuran compound has a structure shown as a formula (I):

wherein:

R₁、R₂、R₃、R₄independently selected from hydrogen, halogen, cyano, nitro, amino, hydroxy, trifluoromethyl, substituted or unsubstituted C_1-6Alkyl, substituted or unsubstituted C_1-6Alkoxy, substituted or unsubstituted C_1-6Alkylamino, substituted or unsubstituted C_3-8Cycloalkyl, substituted or unsubstituted C_3-8Cycloalkoxy, substituted or unsubstituted C_3-8Cycloalkylamino, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted 3-to 8-membered heterocyclyl with 1 to 2 heteroatoms selected from N and O, -COR^a、-CO₂R^a、-CONR^aR^b、-NR^aC(O)R^b、-NR^aSO₂R^b、-SR^a、 -SOR^a、-SO₂R^a、-SO₂NR^aR^b、-OC(O)R^aor-OC (O) NR^aR^b，

R^aAnd R^bIndependently of one another is hydrogen, C_1-6Alkyl radical, C_3-6Cycloalkyl, aryl or heteroaryl;

R₅is hydrogen, halogen, cyano, nitro, amino, hydroxy, trifluoromethyl, substituted or unsubstituted C_1-8Alkyl, substituted or unsubstituted C_1-8Alkoxy, substituted or unsubstituted C_1-8Alkylamino, substituted or unsubstituted C_3-8Cycloalkyl, substituted or substituted C_3-8Cycloalkoxy, substituted or unsubstituted C_1-8Alkylamino, substituted or unsubstituted aryl or heteroaryl;

R₆、R₇、R₈、R₉independently selected from hydrogen, halogen, cyano, nitro, amino, hydroxy, trifluoromethyl, C_1-3Alkoxy radical, C_1-3An alkylamino group.

Through repeated research, the inventor of the invention finds that the obtained nitrofuran compound has the characteristics of drug forming property and the like by constructing a skeleton with the advantage of drug-like property and reasonably substituting, has high selectivity on STAT3 protein, has obvious inhibition effect on STAT3, can be used as a STAT3 specific inhibitor, and has the characteristics of high selectivity, strong drug effect, good drug forming property, safety and the like for inhibiting the STAT3 protein.

In addition, the nitrofuran compound can obviously reduce the volume and weight of tumors in vivo and obviously inhibit the growth and proliferation of the tumors; in addition, when the nitrofuran compound or the pharmaceutically acceptable salt or solvate thereof and the EGFR inhibitor are used together, the generation of acquired drug resistance of the EGFR drug can be delayed, the clinical service life of the EGFR drug can be prolonged, and the nitrofuran compound or the pharmaceutically acceptable salt or solvate thereof has important clinical significance.

Preferably, R₁、R₂、R₃、R₄Is substituted at least at 1 position with the following substituents: halogen, cyano, amino, nitro, hydroxy, trifluoromethyl, C_1-3Alkyl radical, C_1-3Alkoxy radical, C_1-3Alkylamino, 3-to 8-membered heterocyclyl containing 1-2 heteroatoms selected from N and O, -COR^a’、-CO₂R^a’、-CONR^a’R^b’、-NR^a’C(O)R^b’、-NR^a’SO₂R^b’、 -SR^a’、-SOR^a’、-SO₂R^a’、-SO₂NR^a’R^b’、-OC(O)R^a’、-OC(O)NR^a’R^b’。

R^a' and R^b' independently is hydrogen, C_1-6Alkyl radical, C_3-6Cycloalkyl, aryl or heteroaryl;

preferably, R₁、R₂、R₃、R₄Is independently selected fromH. Halogen, cyano, nitro, amino, hydroxy, trifluoromethyl, C_1-6Alkyl radical, C_1-6Heteroalkylalkoxy, C_1-6Alkylamino radical, C_3-8Cycloalkyl radical, C_3-8Cycloalkoxy, C_3-8Cycloalkylamino, aryl, heteroaryl, 3-to 8-membered heterocyclyl containing 1-4 heteroatoms selected from N and O.

More preferably, the 3-to 8-membered heterocyclic group containing 1 to 4 heteroatoms selected from N and O is a group including, but not limited to: 1- (3-oxetanyl) piperazinyl, homopiperazinyl, N-methylpiperazinyl, 1-t-butoxycarbonylpiperazinyl, morpholinyl, 1-thiomorpholinyl or 3, 6-dihydropyridinyl.

Preferably, R₅Is substituted at least at 1 position with the following substituents: halogen, cyano, amino, nitro, hydroxy, trifluoromethyl, methylthio, C_1-3Alkyl radical, C_1-3Alkoxy or C_1-3An alkylamino group.

Preferably, R₁、R₂、R₃、R₄、R₅Wherein said aryl or heteroaryl is independently selected from the group consisting of: furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, benzofuryl, benzothienyl, benzoxazolyl, benzothiazolyl, phenyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, quinolinyl, or naphthyl.

Preferably, the nitrofurans compound has a structure shown in the following number 1-26:

the preparation method of the nitrofuran compound comprises the following steps:

s1: dissolving the formula (1) and the formula (2) in a solvent, and generating an intermediate (3) after reaction;

s2: inserting a side chain into the intermediate (3) to generate an intermediate (4);

s3: reducing the intermediate (4) to generate an intermediate (5), and then carrying out condensation reaction to obtain the nitrofuran compound;

specifically, the reaction process is as follows:

preferably, the solvent in S1 is one or more of acetone, methanol, ethanol, or acetonitrile.

Preferably, the temperature of the reaction in S2 is 40-100 ℃.

Preferably, in S3, side chain insertion into intermediate (3) under the catalysis of palladium catalyst generates intermediate (4).

The application of the nitrofurans or the pharmaceutically acceptable salt or solvate thereof in preparing the medicines for inhibiting the STAT3 protein activity is also within the protection scope of the invention.

Preferably, the pharmaceutically acceptable salt is a pharmaceutically acceptable organic or inorganic salt.

Specifically, pharmaceutically acceptable salts include, but are not limited to: sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, gluconate, formate, benzoate, glutamate, methanesulfonate (methanesulfonate), ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate; or ammonium salts (e.g., primary amine salts, secondary amine salts, tertiary amine salts, quaternary ammonium salts), metal salts (e.g., sodium salts, potassium salts, calcium salts, magnesium salts, manganese salts, iron salts, zinc salts, copper salts, lithium salts, aluminum salts).

Preferably, the nitrofurans compound or the pharmaceutically acceptable salt or solvate thereof is used for preparing a medicament for treating diseases related to abnormal cell proliferation, morphological change and hyperkinesia of STAT3 high expression, or treating diseases related to angiogenesis or cancer metastasis; in particular to the application in preparing the medicine for treating or preventing the growth and the metastasis of the tumor.

Experimental research shows that the nitrofurans compound or pharmaceutically acceptable salts and solvates thereof can obviously inhibit proliferation, migration and invasion of various in vitro tumor cells.

More preferably, the nitrofurans compound or the pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition is used for preparing the drugs for inhibiting the proliferation, migration and invasion of tumor cells.

Further preferably, the nitrofurans compound or the pharmaceutically acceptable salt or solvate thereof is used for preparing the medicine for promoting the apoptosis of tumor cells.

Preferably, cancers include, but are not limited to: acute lymphocytic leukemia, acute myelocytic leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, extrahepatic-biliary cancer, bladder cancer, bone cancer, brain stem glioma, brain tumor, bronchial adenoma, Burkitt's lymphoma, carcinoid tumor, cancer of unknown primary origin, central nervous system lymphoma, cervical cancer, gastric cancer, renal cancer, laryngeal cancer, blood cancer, liver cancer, non-small cell lung cancer, melanoma, prostate cancer, rectal cancer, salivary gland cancer, sarcoma, small intestine cancer, soft tissue sarcoma, uterine sarcoma, testicular cancer, breast cancer, ovarian cancer, rhabdoid tumor, synovial sarcoma, mesothelioma, skin cancer, oral cancer, fallopian tube tumor, peritoneal tumor, glioma, glioblastoma, myeloma.

Preferably, the tumor cell is a breast cancer cell, a lung cancer cell, a gastric adenocarcinoma and/or a gastric cancer cell.

The inventor of the invention researches a STAT3 inhibition mechanism of a nitrofuran compound through a series of experiments, and the results show that the nitrofuran compound can obviously inhibit STAT3 dimerization and combination of STAT3 and DNA, inhibit the tyrosine phosphorylation level of STAT3, inhibit the expression of downstream target genes BCL-XL, C-myc and Mcl-1 of STAT3 and have concentration dependence.

Preferably, the nitrofurans compound or the pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition is used for preparing the medicine for inhibiting the tyrosine phosphorylation level of STAT 3.

The invention also claims the application of the nitrofuran compound or the pharmaceutically acceptable salt or solvate thereof in preparing the medicine for inhibiting the growth and proliferation of the tumor.

The influence of the compound on in-vivo tumors is investigated through in-vivo mouse experiments, and the results show that the compound has no toxicity on visceral organs of mice, and the compound can obviously reduce the volume and weight of in-vivo tumors, namely obviously inhibit the growth and proliferation of the tumors.

The invention also claims a pharmaceutical composition, which comprises the nitrofuran compound or the pharmaceutically acceptable salt or solvate thereof and an EGFR inhibitor.

Preferably, the EGFR inhibitor is one or more of gefitinib, erlotinib hydrochloride, camatinib hydrochloride, afatinib maleate, dacatinib, oxitinib mesylate or acitinib mesylate tablets.

Compared with the prior art, the invention has the following advantages and effects:

the nitrofuran compound provided by the invention has a skeleton with the advantage of drug property, has the characteristics of drug forming property and the like, has high selectivity on STAT3 protein, has a remarkable inhibiting effect on STAT3, and can be used as a STAT3 specific inhibitor; in addition, the nitrofurans compound can obviously reduce the volume and weight of tumors in vivo and obviously inhibit the growth and proliferation of the tumors;

drawings

FIG. 1 is a plot of the growth inhibition of Compound 23 against a variety of cancer cells, as well as IC 50;

figure 2 is a graph of the results of compound 23 inhibiting phosphorylation of STAT3 and dimer formation; FIG (2A) shows the effect of compound 23 treatment on the pY705-STAT3 protein of gastric cancer cells at various time points; FIG (2B) shows the effect of different concentrations of Compound 23 on the pY705-STAT3 protein from gastric cancer cells;

FIG. 3 is a graph showing the results of inhibition of STAT3 transcriptional activity and target gene expression by Compound 23; figure (3A) shows the effect of compound 23 on STAT3 transcriptional activity; figure (3B) shows the effect of compound 23 on target genes downstream of STAT 3.

FIG. 4 is a graph showing the results of Compound 23 in inhibiting gastric cancer tumor growth in vivo; FIG. 4A is a graph showing subcutaneous gastric cancer tumors obtained by sacrifice of mice 21 days after administration to each group of nude mice; FIG. 4B is a graph showing a statistical tumor volume in nude mice during administration to each group of nude mice for 21 days; FIG. 4C is a graph showing subcutaneous gastric cancer tumor weight statistics obtained by obtaining materials from mice sacrificed 21 days after administration to each group of nude mice; FIG. 4D is a graph showing a statistical graph of body weights of nude mice during administration to the nude mice of each group for 21 days;

FIG. 5 is a graph showing the results of compound 23 inhibiting the expression of pY705-STAT3 protein and the target gene in vivo; FIG. 5A shows that the tissue protein of the tumor is extracted and the protein expression of pY705-STAT3 and its downstream target gene is detected after the tumor is stripped from each group of nude mice; FIG. 5B is a graph showing the results of subcutaneous gastric cancer HE staining obtained by obtaining materials from sacrificed mice 21 days after administration to each group of nude mice; FIG. 5C is a graph showing the results of HE staining of heart, liver, spleen, lung and kidney obtained by sacrifice of mice 21 days after administration to each group of nude mice.

Detailed Description

The present invention will be further explained with reference to the following examples and drawings, but the examples are not intended to limit the present invention in any manner. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the present invention are commercially available.

Example 1

The structure and preparation process of (N- (4- (7-methoxyimidazo [1,2-a ] pyridin-3-yl) phenyl) -5-nitrofuran-2-carboxamide (1) are shown as follows:

the preparation process comprises the following steps:

step 1: preparation of 7-methoxyimidazo [1,2-a ] pyridine (1a)

4-methoxypyridin-2-amine (7.37g, 59.4mol) and 2-chloroacetaldehyde (11.64g, 148.6mol) were added to a clean flask, dissolved in 25mL of ethanol and sodium bicarbonate (9.98g, 118.9mol) was added. After stirring at 78 ℃ for 4h at rt, separation was performed by extraction with ethyl acetate and water, the organic phases were combined, dried over anhydrous sodium sulfate, the solvent was dried and column chromatographed (dichloromethane/methanol 30:1, V/V) to give the desired product as a brown oil (9.95g, 87.0%).¹H NMR(400 MHz,Chloroform-d)δ7.91(d,J＝7.6Hz,1H),7.47(d,J＝1.4Hz,1H),7.39(d,J＝1.9Hz,1H), 6.86(d,J＝2.5Hz,1H),6.49(dd,J＝7.4,2.5Hz,1H),3.84(s,3H).MS(EI)m/z 149.01(M+H)⁺.

Step 2: preparation of 7-methoxy-3- (4-nitrophenyl) imidazo [1,2-a ] pyridine (1 b).

1a (700mg, 4.6mmol), potassium carbonate (3.174mg, 23mmol) and 1-bromo-4-nitrobenzene (1.4 g, 7mmol) were charged to a clean flask at room temperature, dissolved in 10mL of N, N-dimethylformamide and the catalyst palladium acetate (51.7mg, 0.23mmol) was added to the reaction under nitrogen. After stirring at 130 ℃ for 12h, the solution was diluted with 100ml of ethyl acetate. The organic layer was washed with brine and dried over anhydrous sodium sulfate. The resulting solution was spun dry and purified by silica gel column chromatography (65: 1, V/V dichloromethane/methanol) to give the desired product (825mg, 67%) as a red-brown solid.¹H NMR(500MHz, Chloroform-d)δ8.36(m,2H),8.24(d,J＝7.4Hz,1H),7.71(m,3H),6.97(d,J＝2.6Hz,1H),6.65 (dd,J＝7.3,2.9Hz,1H).¹³C NMR(126MHz,Chloroform-d)δ158.37,149.20,146.47,136.09, 134.52,126.94,124.78,123.84,122.95,108.62,95.76,55.70；MS(EI)m/z 270.08(M+H)⁺.

And step 3: preparation of 4- (7-Methoxyimidazo [1,2-a ] pyridin-3-yl) aniline (1c)

1b (690mg, 2.57mmol) was dissolved in 15mL ethanol, ammonium chloride (1374mg, 25.7 mmol) was dissolved in 15mL water at room temperature, the two were mixed, and iron powder (1000mg, 17.99mmol) was added thereto. After stirring at 80 ℃ for 4 hours, the solution was filtered through celite to remove iron powder. The resulting solution was evaporated and purified by silica gel column chromatography (dichloromethane/methanol 200:1, V/V) to give the desired product as a tan solid (457mg, 74%).¹H NMR(400MHz, Chloroform-d)δ8.06(d,J＝8.2Hz,1H),7.44(s,1H),7.32–7.28(m,2H),6.91(d,J＝2.5Hz, 1H),6.83–6.78(m,2H),6.51(dd,J＝7.6,2.5Hz,1H),3.88(s,3H)；MS(EI)m/z 240.11(M+H)⁺.

And 4, step 4: preparation of (N- (4- (7-methoxyimidazo [1,2-a ] pyridin-3-yl) phenyl) -5-nitrofuran-2-carboxamide (1)

1c (150mg, 0.62mmol) was dissolved in 15mL of N, N-dimethylformamide and 5-nitrofuran-2-carboxylic acid (147.8mg, 0.94mmol), HATU (509.2mg, 1.34mmol) and N, N-diisopropylethylamine (239.94 mg, 1.84mmol) were added thereto at room temperature. After stirring at room temperature for 4 hours, the solution was diluted with 100ml of ethyl acetate. The organic layer was washed with brine and dried over anhydrous sodium sulfate. The resulting solution was evaporated and purified by silica gel column chromatography (dichloromethane: methanol 75:1, V/V) to give the title product as a tan solid (200mg, 85%);¹H NMR(400MHz,DMSO-d₆)δ 10.88(s,1H),8.60(d,J＝7.6Hz,1H),8.02(s,1H),8.00–7.95(m,2H),7.85(d,J＝3.9Hz,1H), 7.74–7.68(m,3H),7.23(d,J＝2.6Hz,1H),7.01(dd,J＝7.6,2.6Hz,1H),3.98(s,3H).MS(EI) m/z 379.10(M+H)⁺；HRMS(ESI)calcd for C₁₉H₁₄N₄O₅(M+H)⁺:379.1037；found 379.1035.

example 2

The structure and preparation process of N- (3-fluoro-4- (7-methoxyimidazo [1,2-a ] pyridin-3-yl) phenyl) -5-nitrofuran-2-carboxamide (2) are shown as follows:

the preparation process comprises the following steps:

step 1: preparation of 3- (2-fluoro-4-nitrophenyl) -7-methoxyimidazo [1,2-a ] pyridine (2b)

The 1-bromo-4-nitrobenzene was replaced with 1-bromo-2-fluoro-4-nitrobenzene and the remaining required starting materials, reagents and preparation were the same as in step 2 of example 1 to give a tan solid.

¹H NMR(400MHz,Chlooform-d)δ8.19(dd,J＝8.5,2.3Hz,1H),8.14(dd,J＝10.0,2.3Hz, 1H),7.94(dd,J＝7.5,3.2Hz,1H),7.80–7.69(m,2H),7.02(d,J＝2.5Hz,1H),6.68(dd,J＝7.6, 2.5Hz,1H),3.93(s,3H)；MS(EI)m/z 288.07(M+H)⁺.

Step 2: 3-fluoro-4- (7-methoxyimidazo [1,2-a ] pyridin-3-yl) aniline (2c)

The 1b was replaced with 2b and the remaining required starting materials, reagents and preparation were the same as in example 1, step 3, giving a tan solid.¹H NMR(400MHz,DMSO-d₆)δ7.91(dd,J＝7.5,2.8Hz,1H),7.38(s,1H),7.15(t,J＝8.6 Hz,1H),6.99(d,J＝2.5Hz,1H),6.63(dd,J＝7.5,2.5Hz,1H),6.55–6.47(m,2H),5.74(s,2H), 3.85(s,3H).¹³C NMR(126MHz,DMSO-d₆)δ161.91,159.98,157.49,151.96,132.31,132.06, 125.80,119.85,110.83,107.30,102.83,100.76,95.24,56.08；MS(EI)m/z 2548.10(M+H)⁺.

And step 3: preparation of N- (3-fluoro-4- (7-methoxyimidazo [1,2-a ] pyridin-3-yl) phenyl) -5-nitrofuran-2-carboxamide (2).

The 1c was replaced with 2c and the remaining required starting materials, reagents and preparation were the same as in step 4 of example 1 to give a tan solid.¹H NMR(400MHz,DMSO-d₆)δ11.04(s,1H),8.27(dd,J＝7.6,2.8Hz,1H),7.95(dd,J＝ 12.7,2.0Hz,1H),7.89–7.83(m,2H),7.76(dd,J＝8.5,2.0Hz,1H),7.72–7.65(m,2H),7.17(d, J＝2.5Hz,1H),6.89(dd,J＝7.5,2.5Hz,1H),3.94(s,3H)；MS(EI)m/z 397.09(M+H)⁺；HRMS (ESI)calcd for C₁₉H₁₃N₄O₅F(M+H)⁺:397.0943；found 397.0941.

Example 3

The structure and preparation process of N- (3-fluoro-4- (7-methoxyimidazo [1,2-a ] pyridin-3-yl) phenyl) -5-nitrofuran-2-carboxamide (3) are shown as follows:

the preparation process comprises the following steps:

step 1: preparation of 7-methoxy-3- (2-methyl-4-nitrophenyl) imidazo [1,2-a ] pyridine (3b)

The 1-bromo-4-nitrobenzene was replaced with 1-bromo-2-methyl-4-nitrobenzene and the remaining required starting materials, reagents and preparation were the same as in example 1, step 2, to give a tan solid.

¹H NMR(400MHz,Chloroform-d)δ8.25(d,J＝2.4Hz,1H),8.16(dd,J＝8.4,2.4Hz,1H), 7.64(d,J＝7.4Hz,1H),7.54(m,2H),6.97(d,J＝2.5Hz,1H),6.58(dd,J＝7.5,2.5Hz,1H),3.90 (s,3H),2.36(s,3H)；MS(EI)m/z 284.10(M+H)⁺.

Step 2: preparation of 4- (7-Methoxyimidazo [1,2-a ] pyridin-3-yl) -3-methylaniline (3c)

The 1b was replaced with 3b and the remaining required starting materials, reagents and preparation were the same as in example 1, step 3, giving a tan solid.¹H NMR(400MHz,Chloroform-d)δ7.56(d,J＝7.5Hz,1H),7.36(s,1H),7.09(d,J＝8.1 Hz,1H),6.91(d,J＝2.5Hz,1H),6.67(d,J＝2.4Hz,1H),6.61(dd,J＝8.1,2.5Hz,1H),6.46(dd, J＝7.5,2.5Hz,1H),3.87(s,3H),2.05(s,3H).¹³C NMR(101MHz,Chloroform-d)δ157.48, 147.22,146.52,139.65,132.47,131.70,124.22,123.79,117.98,116.71,112.71,107.09,94.90, 55.52,19.83；MS(EI)m/z 254.12(M+H)⁺.

And step 3: n- (3-fluoro-4- (7-methoxyimidazo [1,2-a ] pyridin-3-yl) phenyl) -5-nitrofuran-2-carboxamide (3)

The 1c was replaced with 3c and the remaining required starting materials, reagents and preparation were the same as in step 4 of example 1 to give a tan solid.

¹H NMR(500MHz,Chloroform-d)δ8.98(s,1H),7.75(d,J＝2.3Hz,1H),7.67(dd,J＝8.2, 2.3Hz,1H),7.60(d,J＝7.5Hz,1H),7.47–7.41(m,3H),7.36(d,J＝8.3Hz,1H),6.92(d,J＝2.5 Hz,1H),6.52(dd,J＝7.5,2.5Hz,1H),3.87(s,3H),2.19(s,3H).¹³C NMR(101MHz, Chloroform-d)δ157.80,154.23,147.88,146.94,139.55,137.13,132.16,131.96,125.41,124.11, 122.79,122.39,118.27,116.99,112.75,107.60,95.01,55.59,20.09；MS(EI)m/z 393.11(M+H)⁺； HRMS(ESI)calcd for C₂₀H₁₆N₄O₅(M+H)⁺:393.1193；found 393.1190.

Example 4

The structure and preparation process of N- (2-methoxy-4- (7-methoxyimidazo [1,2-a ] pyridine-3-yl) phenyl) -5-nitrofuran-2-carboxamide (4) are shown in the following

The preparation process comprises the following steps:

step 1: preparation of 7-methyl-2-phenylimidazo [1,2-a ] pyridine (4b)

The 1-bromo-4-nitrobenzene was replaced with 4-bromo-2-methoxy-1-nitrobenzene, and the remaining required starting materials, reagents and preparation were the same as in step 2 of example 1 to give a yellow solid.¹H NMR(400MHz,Chloroform-d)δ8.20(d,J＝7.5Hz, 1H),8.02(d,J＝8.8Hz,1H),7.67(s,1H),7.21–7.18(m,2H),6.95(d,J＝2.6Hz,1H),6.63(dd,J ＝7.6,2.6Hz,1H),4.02(s,3H),3.90(s,3H)；MS(EI)m/z 300.09(M+H)⁺.

Step 2: preparation of 2-methoxy-4- (7-methoxyimidazo [1,2-a ] pyridin-3-yl) aniline (4c)

The procedure of example 1 was followed by changing 1b to 4b, and the remaining required starting materials, reagents and preparation were the same as in example 1, step 3, to give a brownish yellow solid.

¹H NMR(400MHz,Chloroform-d)δ8.08(dd,J＝7.5,0.7Hz,1H),7.43(s,1H),6.94(dd,J＝ 7.9,1.8Hz,1H),6.91–6.87(m,2H),6.81(d,J＝7.9Hz,1H),6.50(dd,J＝7.5,2.5Hz,1H),3.89 (s,3H),3.87(s,3H).¹³C NMR(101MHz,Chloroform-d)δ157.42,147.53,146.96,136.42,130.66, 125.42,124.05,121.17,119.09,114.98,110.77,107.32,95.02,55.64,55.53；MS(EI)m/z 270.12 (M+H)⁺.

And step 3: preparation of N- (2-methoxy-4- (7-methoxyimidazo [1,2-a ] pyridin-3-yl) phenyl) -5-nitrofuran-2-carboxamide (4)

The 1c was replaced with 4c and the remaining required starting materials, reagents and preparation were the same as in 4 of example 1 to give a tan solid.

¹H NMR(400MHz,Chloroform-d)δ8.92(s,1H),8.55(d,J＝8.3Hz,1H),8.17(d,J＝7.6 Hz,1H),7.56(s,1H),7.43(d,J＝3.8Hz,1H),7.39(d,J＝3.8Hz,1H),7.19(dd,J＝8.3,1.8Hz, 1H),7.07(d,J＝1.8Hz,1H),6.94(d,J＝2.5Hz,1H),6.57(dd,J＝7.6,2.5Hz,1H),4.04(s,3H), 3.90(s,3H).¹³C NMR(126MHz,Chloroform-d)δ157.80,153.79,151.36,148.98,147.97,147.73, 131.79,126.31,125.94,124.46,123.96,120.74,120.23,116.69,112.65,110.04,107.88,95.27, 56.24,55.60；MS(EI)m/z 409.11(M+H)⁺；HRMS(ESI)calcd for C₂₀H₁₆N₄O₆(M+H)⁺:409.1143； found 409.1148.

Example 5

The structure and preparation process of N- (4- (7-methoxyimidazo [1,2-a ] pyridine-3-yl) -2-methylphenyl) -5-nitrofuran-2-formamide (5) are shown in the following

The preparation process comprises the following steps:

step 1: preparation of 7-methoxy-2-phenylimidazo [1,2-a ] pyridine (5b)

The 1-bromo-4-nitrobenzene was changed to 4-bromo-2-methyl-1-nitrobenzene and the remaining required starting materials, reagents and preparation were the same as in example 1, step 2, giving a tan solid.

¹H NMR(400MHz,Chloroform-d)δ8.21(d,J＝7.5Hz,1H),8.15(d,J＝9.1Hz,1H),7.67 (s,1H),7.54–7.47(m,2H),6.96(d,J＝2.6Hz,1H),6.63(dd,J＝7.6,2.6Hz,1H),3.91(s,3H), 2.71(s,3H).¹³C NMR(101MHz,Chloroform-d)δ158.24,148.90,147.51,135.32,134.57,134.00, 130.70,126.09,124.77,123.89,122.89,108.47,95.63,55.68,21.07；MS(EI)m/z 248.10(M+H)⁺.

Step 2: preparation of 4- (7-Methoxyimidazo [1,2-a ] pyridin-3-yl) -2-methylaniline (5c)

The remaining required starting materials, reagents and procedures were the same as in example 1, step 3, replacing 1b with 5b, to give a tan solid.¹H NMR(500MHz,Chloroform-d)δ8.06(d,J＝7.6Hz,1H),7.41(s,1H),7.21–7.10(m, 2H),6.89(d,J＝2.6Hz,1H),6.77(d,J＝8.0Hz,1H),6.48(dd,J＝7.5,2.5Hz,1H),3.85(s,3H), 2.22(s,3H).¹³C NMR(126MHz,Chloroform-d)δ157.39,146.90,144.82,130.49,130.47,127.07, 125.31,124.08,122.85,119.06,115.24,107.22,94.96,55.50,17.43；MS(EI)m/z 254.12(M+H)⁺.

And step 3: n- (4- (7-methoxyimidazo [1,2-a ] pyridin-3-yl) -2-methylphenyl) -5-nitrofuran-2-carboxamide (5)

The 1c was replaced with 5c and the remaining required starting materials, reagents and preparation were the same as in 4 of example 1 to give a tan solid.¹H NMR(400MHz,Chloroform-d)δ8.29(s,1H),8.20(s,1H),8.08(d,J＝8.2Hz,1H), 7.46(m,5H),7.28(s,2H),6.96(s,1H),6.59(d,J＝7.6Hz,1H),3.91(s,3H),2.48(s,3H)；MS(EI) m/z 393.11(M+H)⁺；HRMS(ESI)calcd for C₂₀H₁₆N₄O₅(M+H)⁺:393.1193；found 393.1195.

Example 6

The structure and preparation process of N- (3-methoxy-4- (7-methoxyimidazo [1,2-a ] pyridin-3-yl) phenyl) -5-nitrofuran-2-carboxamide (6) are shown in the following

The preparation process comprises the following steps:

step 1: preparation of 7-methoxy-3- (2-methoxy-4-nitrophenyl) imidazo [1,2-a ] pyridine (6b)

The 1-bromo-4-nitrobenzene was replaced with 1-bromo-2-methoxy-4-nitrobenzene, and the remaining required starting materials, reagents and preparation were the same as in step 2 of example 1 to give a tan solid.

¹H NMR(400MHz,Chloroform-d)δ7.97(dd,J＝8.3,2.2Hz,1H),7.89(d,J＝2.2Hz,1H), 7.75(s,1H),7.58(d,J＝8.4Hz,1H),6.97(s,1H),6.57(d,J＝7.5Hz,1H),3.95(s,3H),3.90(s, 3H)；MS(EI)m/z 300.09(M+H)⁺.

Step 2: preparation of 3-methoxy-4- (7-methoxyimidazo [1,2-a ] pyridin-3-yl) aniline (6c)

The remaining required starting materials, reagents and procedures were the same as in example 1, step 3, replacing 1b with 6b, to give a tan solid.¹H NMR(400MHz,Chloroform-d)δ7.64(dd,J＝7.5,0.7Hz,1H),7.41(s,1H),7.15(d,J＝ 7.9Hz,1H),6.91(d,J＝2.5Hz,1H),6.47(dd,J＝7.5,2.5Hz,1H),6.40–6.35(m,2H),3.88(s, 3H),3.74(s,3H).¹³C NMR(126MHz,Chloroform-d)δ158.33,157.37,148.75,146.89,132.95, 131.59,125.74,122.47,107.95,107.37,106.29,98.35,94.75,55.46,55.30；MS(EI)m/z 270.12 (M+H)⁺.

And step 3: preparation of N- (3-methoxy-4- (7-methoxyimidazo [1,2-a ] pyridin-3-yl) phenyl) -5-nitrofuran-2-carboxamide (6)

The 1c was replaced by 6c and the remaining required starting materials, reagents and preparation were the same as in step 4 of example 1 to give a tan solid.¹H NMR(500MHz,DMSO-d₆)δ10.82(s,1H),7.91–7.81(m,2H),7.71–7.65(m,2H),7.57 (d,J＝8.2Hz,1H),7.48(s,1H),7.41(d,J＝8.1Hz,1H),7.02(s,1H),6.63(d,J＝7.0Hz,1H), 3.87(s,3H),3.80(s,3H)；MS(EI)m/z 409.11(M+H)⁺；HRMS(ESI)calcd for C₂₀H₁₆N₄O₆(M+H)⁺:409.1143；found 409.1141.

Example 7

The structure and preparation process of N- (5- (7-methoxyimidazo [1,2-a ] pyridin-3-yl) pyridin-2-yl) -5-nitrofuran-2-carboxamide (7) are shown as follows:

the preparation process comprises the following steps:

step 1: preparation of 5- (7-Methoxyimidazo [1,2-a ] pyridin-3-yl) pyridin-2-amine (7c)

The 1-bromo-4-nitrobenzene was replaced with 5-bromopyridin-2-amine and the remaining required starting materials, reagents and preparation were the same as in example 1, step 2, giving a tan solid.¹H NMR(500MHz,Chloroform-d)δ8.23(s,1H),7.96(dd,J＝7.4, 2.3Hz,1H),7.56(d,J＝8.5Hz,1H),7.46(s,1H),6.91(d,J＝2.5Hz,1H),6.63(dd,J＝8.5,2.2 Hz,1H),6.53(dd,J＝7.4,2.7Hz,1H),4.67(s,2H),3.88(s,3H).¹³C NMR(126MHz, Chloroform-d)δ158.07,157.61,147.94,147.46,137.95,131.51,123.66,122.00,115.62,108.71, 107.66,95.20,55.55；MS(EI)m/z 241.10(M+H)⁺.

Step 2: preparation of N- (5- (7-methoxyimidazo [1,2-a ] pyridin-3-yl) pyridin-2-yl) -5-nitrofuran-2-carboxamide (7)

The 1c was replaced with 7c and the remaining required starting materials, reagents and preparation were the same as in step 4 of example 1 to give a tan solid.¹H NMR(400MHz,DMSO-d₆)δ11.48(s,1H),8.69(d,J＝2.4Hz,1H),8.52(d,J＝7.6 Hz,1H),8.31(d,J＝8.6Hz,1H),8.19(dd,J＝8.7,2.4Hz,1H),7.93(d,J＝3.9Hz,1H),7.90– 7.69(m,2H),7.12(d,J＝2.6Hz,1H),6.82(dd,J＝7.6,2.5Hz,1H),3.92(s,3H).¹³C NMR(101 MHz,DMSO-d₆)δ159.45,155.59,152.64,151.14,147.51,147.44,137.78,129.79,126.39,121.82, 121.38,117.74,115.26,113.75,108.87,94.60,56.53；MS(EI)m/z 380.09(M+H)⁺；HRMS(ESI) calcd for C₁₈H₁₃N₅O₅(M+H)⁺:380.0989；found 380.0989.

Example 8

The structure and preparation process of N- (3- (7-methoxyimidazo [1,2-a ] pyridine-3-yl) phenyl) -5-nitrofuran-2-formamide (8) are as follows:

the preparation process comprises the following steps:

step 1: preparation of 3- (7-Methoxyimidazo [1,2-a ] pyridin-3-yl) aniline (8c)

The 1-bromo-4-nitrobenzene was replaced with 3-bromoaniline and the remaining required starting materials, reagents and preparation were the same as in step 2 of example 1 to give a tan solid.¹H NMR(500MHz,Chloroform-d)δ8.18(d,J＝7.5Hz,1H),7.50(d, J＝2.3Hz,1H),7.29–7.26(m,1H),6.90(t,J＝3.9Hz,2H),6.82(s,1H),6.71(d,J＝7.6Hz,1H), 6.51(dd,J＝7.5,2.8Hz,1H),3.87(d,J＝2.3Hz,3H)；MS(EI)m/z 240.11(M+H)⁺.

Step 2: preparation of N- (3- (7-methoxyimidazo [1,2-a ] pyridin-3-yl) phenyl) -5-nitrofuran-2-carboxamide (8)

The 1c was replaced with 8c and the remaining required starting materials, reagents and preparation were the same as in step 4 of example 1 to give a tan solid.¹H NMR(400MHz,DMSO-d₆)δ10.86(s,1H),8.59(d,J＝7.6Hz,1H),8.08(d,J＝3.9 Hz,1H),8.03(s,1H),7.86(m,2H),7.67(d,J＝3.9Hz,1H),7.62(t,J＝8.0Hz,1H),7.47(d,J＝ 7.9Hz,1H),7.22(d,J＝2.6Hz,1H),7.04(dd,J＝7.6,2.6Hz,1H),3.98(s,3H).¹³C NMR(101 MHz,DMSO-d₆)δ161.43,155.32,152.31,148.14,144.56,139.11,130.51,127.61,127.05,125.44, 124.87,121.62,120.50,117.37,113.99,110.25,93.39,57.02；MS(EI)m/z 379.10(M+H)⁺；HRMS (ESI)calcd for C₁₉H₁₄N₄O₅(M+H)⁺:379.1037；found 379.1035.

Example 9

The structure and preparation process of N- (4- (7-methoxyimidazo [1,2-a ] pyridin-3-yl) -3- (trifluoromethyl) phenyl) -5-nitrofuran-2-carboxamide (9) are shown as follows:

the preparation process comprises the following steps:

step 1: preparation of 4- (7-Methoxyimidazo [1,2-a ] pyridin-3-yl) -3- (trifluoromethyl) aniline (9c)

The 1-bromo-4-nitrobenzene was replaced with 4-bromo-3- (trifluoromethyl) aniline and the remaining required starting materials, reagents and preparation were the same as in example 1, step 2, giving a tan solid.¹H NMR(500MHz,Chloroform-d)δ7.52(d,J＝7.5 Hz,1H),7.41(s,1H),7.18(dd,J＝8.3,2.6Hz,1H),7.09(d,J＝2.2Hz,1H),6.88(dt,J＝8.7,2.3 Hz,2H),6.45(dt,J＝7.6,1.9Hz,1H),4.18(s,2H),3.86(d,J＝2.4Hz,3H).MS(EI)m/z 308.09 (M+H)⁺.

Step 2: preparation of N- (4- (7-methoxyimidazo [1,2-a ] pyridin-3-yl) -3- (trifluoromethyl) phenyl) -5-nitrofuran-2-carboxamide (9)

The 1c was replaced with 9c and the remaining required starting materials, reagents and preparation were the same as in step 4 of example 1 to give a tan solid.¹H NMR(500MHz,Acetone-d₆)δ10.75(s,1H),8.48(d,J＝2.3Hz,1H),8.24(dd,J＝8.4,2.2 Hz,1H),7.95(d,J＝7.5Hz,1H),7.69–7.61(m,3H),7.55(d,J＝3.9Hz,1H),7.06(d,J＝2.6Hz, 1H),6.71(dd,J＝7.6,2.4Hz,1H),5.05(s,1H),3.91(s,3H)；MS(EI)m/z 447.08(M+H)⁺； HRMS(ESI)calcd for C₂₀H₁₃N₄O₅F₃(M+H)⁺:447.0911；found 447.0912.

Example 10

The structure and preparation process of N- (4- (7-methoxyimidazo [1,2-a ] pyridin-3-yl) -2- (trifluoromethyl) phenyl) -5-nitrofuran-2-carboxamide (10) are shown below:

the preparation process comprises the following steps:

step 1: preparation of 4- (7-Methoxyimidazo [1,2-a ] pyridin-3-yl) -2- (trifluoromethyl) aniline (10c)

1-bromo-4-nitrobenzene is changed into 4-iodo-2- (trifluoromethyl) aniline,the remaining required starting materials, reagents and preparation were the same as in step 2 of example 1 to give a tan solid.¹H NMR(500MHz,Methanol-d4)δ7.94(d,J＝7.6Hz, 1H),7.36(d,J＝2.2Hz,1H),7.28–7.23(m,2H),6.88(d,J＝8.5Hz,1H),6.78(d,J＝2.6Hz, 1H),6.48(dd,J＝7.5,2.5Hz,1H),3.74(s,3H).MS(EI)m/z 308.09(M+H)⁺.

Step 2: preparation of N- (4- (7-methoxyimidazo [1,2-a ] pyridin-3-yl) -2- (trifluoromethyl) phenyl) -5-nitrofuran-2-carboxamide (10)

The 1c was replaced with 10c and the remaining required starting materials, reagents and preparation were the same as in step 4 of example 1 to give a tan solid.¹H NMR(400MHz,DMSO-d₆)δ10.70(s,1H),8.53(d,J＝7.5Hz,1H),8.05–7.99(m,2H), 7.84(d,J＝3.9Hz,1H),7.80(s,1H),7.69(d,J＝8.1Hz,1H),7.65(d,J＝3.9Hz,1H),7.08(d,J＝ 2.6Hz,1H),6.72(dd,J＝7.5,2.6Hz,1H),3.88(s,3H)；HRMS(ESI)calcd for C₂₀H₁₃N₄O₅F₃ (M+H)⁺:447.0911；found 447.0912.

Example 11

The structure and preparation process of N- (4- (7-bromoimidazo [1,2-a ] pyridin-3-yl) phenyl) -5-nitrofuran-2-carboxamide (11) are as follows:

the preparation process comprises the following steps:

step 1: preparation of 7-bromoimidazo [1,2-a ] pyridine (11a)

The 4-methoxypyridin-2-amine was replaced with 4-bromopyridin-2-amine and the remaining required starting materials, reagents and preparation were the same as in example 1, step 1 to give a brown oil.¹H NMR(400MHz,Chloroform-d)δ7.87(d,J＝7.2Hz,1H), 7.67(d,J＝2.0Hz,1H),7.46(d,J＝12.8Hz,2H),6.73(dd,J＝7.2,2.0Hz,1H).¹³C NMR(126 MHz,Chloroform-d)δ145.38,134.10,125.99,119.80,118.03,116.10,112.68；MS(EI)m/z 196.96 (M+H)⁺.

Step 2: preparation of 7-bromo-3- (4-nitrophenyl) imidazo [1,2-a ] pyridine (11b)

Changing 1a to 11a, changing 1-bromo-4-nitrobenzene to 1-iodo-4-nitrobenzene, and the remaining required raw materials, reagents and preparation method were the same as in step 2 of example 1 to give a yellow solid.¹H NMR(400MHz,DMSO-d₆)δ8.68(dd,J＝7.3, 0.8Hz,1H),8.40–8.32(m,2H),8.11–8.03(m,2H),8.02–7.97(m,2H),7.20(dd,J＝7.3,2.1 Hz,1H).¹³C NMR(126MHz,DMSO)δ147.34,146.67,136.07,135.54,128.18,126.26,124.93, 124.38,120.15,119.18,117.27；MS(EI)m/z 317.98(M+H)⁺.

And step 3: preparation of 4- (7-bromoimidazo [1,2-a ] pyridin-3-yl) aniline (11c)

The remaining required starting materials, reagents and preparation were the same as in example 1, step 3, replacing 1b with 11b, to give a white solid.¹H NMR(500MHz,Chloroform-d)δ8.11(d,J＝7.3Hz,1H),7.83(s,1H),7.60(s,1H), 7.29(m,2H),6.86(d,J＝7.3Hz,1H),6.80(d,J＝8.0Hz,2H).¹³C NMR(126MHz,Chloroform-d) δ147.00,132.01,129.68,129.59,123.87,120.18,118.20,117.54,116.15,115.49,113.89.MS(EI) m/z 288.00(M+H)⁺.

And 4, step 4: preparation of N- (4- (7-bromoimidazo [1,2-a ] pyridin-3-yl) phenyl) -5-nitrofuran-2-carboxamide (11)

The 1c was replaced with 11c and the remaining required starting materials, reagents and preparation were the same as in step 4 of example 1 to give a tan solid.¹H NMR(400MHz,DMSO-d₆)δ11.01(s,1H),8.54(d,J＝7.4Hz,1H),7.99(dd,J＝9.3, 2.3Hz,3H),7.84(s,2H),7.79(s,1H),7.73–7.66(m,2H),7.10(dd,J＝7.3,2.0Hz,1H).¹³C NMR(126MHz,DMSO-d₆)δ155.17,152.30,148.25,138.37,132.92,130.11,128.62,125.87, 124.61,121.65,119.68,118.31,117.27,116.84,113.96.MS(EI)m/z 427.00(M+H)⁺；HRMS(ESI) calcd for C₁₈H₁₁N₄O₄Br(M+H)⁺:427.0036；found 427.0033.

Example 12

The structure and preparation process of N- (4- (7-chloro imidazo [1,2-a ] pyridine-3-yl) phenyl) -5-nitrofuran-2-formamide (12) are as follows:

the preparation process comprises the following steps:

step 1: preparation of 7-chloroimidazo [1,2-a ] pyridine (12a)

The 4-methoxypyridin-2-amine was replaced with 4-chloropyridin-2-amine, and the remaining required starting materials, reagents and preparation were the same as in example 1, step 1 to give a brown oil.¹H NMR(400MHz,CDCl₃)δ8.07,8.06,7.64,7.64,7.64,7.58, 7.57,6.80,6.80,6.79,6.78.¹³C NMR(126MHz,CDCl₃)δ145.20,134.46,130.85,125.96,116.73, 114.05,112.57；MS(EI)m/z 153.58(M+H)⁺.

Step 2: preparation of 7-chloro-3- (4-nitrophenyl) imidazo [1,2-a ] pyridine (12b)

The remaining required starting materials, reagents and preparation were the same as in example 1, step 2, replacing 1a with 12a, to give a yellow solid.¹H NMR(400MHz,DMSO-d₆)δ8.74(dd,J＝7.3,0.8Hz,1H),8.39–8.34(m,2H),8.07 (s,1H),8.03–7.98(m,2H),7.91(dd,J＝2.3,0.8Hz,1H),7.12(dd,J＝7.4,2.2Hz,1H).¹³C NMR (126MHz,DMSO-d₆)δ147.01,146.68,136.24,135.55,131.26,128.19,126.38,124.94,124.38, 116.90,114.96；MS(EI)m/z 274.03(M+H)⁺..

And step 3: preparation of 4- (7-chloroimidazo [1,2-a ] pyridin-3-yl) aniline (12c)

The other required starting materials, reagents and preparation were the same as in example 1, step 3, replacing 1b with 12b, to give a white solid.¹H NMR(400MHz,Chloroform-d)δ8.15(dd,J＝7.3,0.8Hz,1H),7.63(dd,J＝2.1,0.8Hz, 1H),7.57(s,1H),7.35–7.27(m,2H),6.85–6.78(m,2H),6.75(dd,J＝7.4,2.1Hz,1H).¹³C NMR(126MHz,Chloroform-d)δ147.02,145.14,131.90,130.49,129.63,126.46,123.82,118.10, 116.74,115.48,113.98；MS(EI)m/z 244.05(M+H)⁺.

And 4, step 4: preparation of N- (4- (7-chloroimidazo [1,2-a ] pyridin-3-yl) phenyl) -5-nitrofuran-2-carboxamide (12)

The 1c was replaced with 12c and the remaining required starting materials, reagents and preparation were the same as in step 4 of example 1 to give a tan solid.¹H NMR(500MHz,DMSO-d₆)δ10.83(s,1H),8.60(dd,J＝7.4,1.8Hz,1H),7.95(d,J＝ 8.3Hz,2H),7.86–7.83(m,2H),7.81(d,J＝1.5Hz,1H),7.70(dt,J＝12.1,3.1Hz,3H),7.02(dd, J＝7.4,2.1Hz,1H).¹³C NMR(126MHz,DMSO-d₆)δ155.16,152.30,148.26,138.25,133.69, 129.99,128.56,125.84,124.82,121.66,117.24,116.64,114.31,113.97；MS(EI)m/z 383.05 (M+H)⁺；HRMS(ESI)calcd for C₁₈H₁₁N₄O₄Cl(M+H)⁺:383.0542；found 383.0539.

Example 13

The structure and preparation process of N- (4- (7- (1-methylpiperidin-4-yl) imidazo [1,2-a ] pyridin-3-yl) phenyl) -5-nitrofuran-2-carboxamide (13) are as follows:

the preparation process comprises the following steps:

step 1: preparation of 7- (4-methylpiperazin-1-yl) -3- (4-nitrophenyl) imidazo [1,2-a ] pyridine (13b)

11b (400mg, 1.26mmol) and 1-methylpiperazine (151mg, 1.51mmol) were added to a clean flask and dissolved in 10mL of N, N-dimethylformamide, and cesium carbonate (821mg, 2.52mmol), BINAP (235.11 mg, 0.378mmol) and Pd (dba)3(115.29mg, 0.126mmol) were added to the flask under nitrogen at room temperature. After stirring at 110 ℃ for 12h, the reaction solution was diluted with 100ml of ethyl acetate. The organic layer was washed with brine and dried over anhydrous sodium sulfate. The resulting solution was evaporated and purified by silica gel column chromatography (dichloromethane: methanol 10:1, V/V) to give the target product as a red brown solid (230mg, 54%).¹H NMR(500MHz,Chloroform-d)δ8.33(d,J＝8.5Hz,2H),8.24 (d,J＝7.7Hz,1H),7.72–7.66(m,3H),6.87(d,J＝2.5Hz,1H),6.72(dd,J＝7.8,2.5Hz,1H), 3.33(t,J＝5.1Hz,4H),2.60(t,J＝5.1Hz,4H),2.37(s,3H).¹³C NMR(126MHz,Chloroform-d) δ149.78,148.99,146.04,136.32,134.80,126.34,124.77,123.46,122.26,107.04,97.94,54.54, 47.97,46.08；MS(EI)m/z 338.15(M+H)⁺.

Step 2: preparation of 4- (7- (4-methylpiperazin-1-yl) imidazo [1,2-a ] pyridin-3-yl) aniline (13c)

The remaining required starting materials, reagents and preparation were the same as in example 1, step 3, replacing 1b with 13b, to give a tan solid.¹H NMR(500MHz,Chloroform-d)δ8.04(d,J＝7.7Hz,1H),7.40(s,1H),7.27(d,J＝ 8.3Hz,2H),6.86–6.75(m,3H),6.57(dd,J＝7.7,2.5Hz,1H),3.28–3.22(m,4H),2.58(t,J＝ 5.0Hz,4H).¹³C NMR(126MHz,CDCl₃)δ148.09,147.42,146.35,130.65,129.26,124.55, 123.49,119.39,115.45,106.38,98.15,54.68,48.51,46.10；MS(EI)m/z 308.17(M+H)⁺.

And step 3: preparation of N- (4- (7- (1-methylpiperidin-4-yl) imidazo [1,2-a ] pyridin-3-yl) phenyl) -5-nitrofuran-2-carboxamide (13)

The 1c was replaced with 13c and the remaining required starting materials, reagents and preparation were the same as in step 4 of example 1 to give a tan solid.¹H NMR(500MHz,Chloroform-d)δ8.90(s,1H),8.14(d,J＝7.6Hz,1H),7.85(d,J＝ 8.1Hz,2H),7.56–7.49(m,3H),7.43(q,J＝3.7Hz,2H),6.84(s,1H),6.65(dd,J＝7.7,2.3Hz, 1H),3.29(t,J＝5.1Hz,4H),2.59(t,J＝5.0Hz,4H),2.36(s,3H).¹³C NMR(126MHz, Chloroform-d)δ154.18,151.25,148.64,147.85,136.06,131.22,128.29,126.53,123.59,123.50, 121.25,116.97,112.72,106.74,97.59,54.60,48.22,46.09；MS(EI)m/z 447.17(M+H)⁺；HRMS (ESI)calcd for C₁₉H₁₃N₄O₅F(M+H)⁺:447.1775；found 447.1773.

Example 14

The structure and preparation process of 5-nitro-N- (4- (7- (pyrrolidin-1-yl) imidazo [1,2-a ] pyridin-3-yl) phenyl) furan-2-carboxamide (14) are as follows:

the preparation process comprises the following steps:

step 1: preparation of 3- (4-Nitrophenyl) -7- (pyrrolidin-1-yl) imidazo [1,2-a ] pyridine (14b)

The 1-methylpiperazine was replaced by pyrrolidine, and the remaining required starting materials, reagents and preparation were the same as in step 1 of example 13 to give a tan solid.¹H NMR(500MHz,Chloroform-d)δ8.33–8.27(m,2H),8.22(d,J＝7.4Hz, 1H),7.68–7.63(m,3H),6.52–6.45(m,2H),3.43–3.36(m,4H),2.11–2.05(m,4H).¹³C NMR (126MHz,Chloroform-d)δ150.74,145.75,145.52,136.71,134.94,125.66,124.78,123.58, 121.36,105.00,92.62,47.76,25.45；MS(EI)m/z 309.12(M+H)⁺.

Step 2: preparation of 4- (7- (pyrrolidin-1-yl) imidazo [1,2-a ] pyridin-3-yl) aniline (14c)

The procedure of example 1 was followed by changing 1b to 14b and the remaining required starting materials, reagents and preparation to give a tan solid.¹H NMR(400MHz,Chloroform-d)δ8.02(d,J＝7.6Hz,1H),7.31(d,J＝12.5Hz,2H), 7.28(s,1H),6.86–6.72(m,2H),6.50(d,J＝2.4Hz,1H),6.40(dd,J＝7.6,2.4Hz,1H),3.41– 3.34(m,4H),2.08–2.03(m,4H).¹³C NMR(126MHz,Chloroform-d)δ147.34,146.42,145.61, 129.36,127.57,123.73,123.62,118.99,115.48,104.49,91.27,47.83,25.42；MS(EI)m/z 279.15 (M+H)⁺.

And step 3: preparation of 5-nitro-N- (4- (7- (pyrrolidin-1-yl) imidazo [1,2-a ] pyridin-3-yl) phenyl) furan-2-carboxamide (14)

The procedure of example 1 was followed by changing 1c to 14c and the remaining required starting materials, reagents and preparation to give a tan solid.¹H NMR(500MHz,DMSO-d₆)δ10.89(s,1H),8.45(d,J＝7.7Hz,1H),8.03–7.96(m, 2H),7.93(s,1H),7.85(d,J＝3.8Hz,1H),7.76–7.66(m,3H),6.91(dd,J＝7.8,2.4Hz,1H),6.46 (d,J＝2.4Hz,1H),3.46(s,4H),2.07–1.99(m,4H).¹³C NMR(126MHz,DMSO-d₆)δ155.29, 152.34,149.49,148.11,142.96,139.41,129.66,127.10,124.51,122.09,121.59,118.37,117.42, 113.98,107.79,85.90,48.38,25.38；MS(EI)m/z 418.1510(M+H)⁺；HRMS(ESI)calcd for C₂₂H₁₉N₅O₄(M+H)⁺:418.1510；found 418.1510.

Example 15

The structure and preparation process of N- (4- (7- (1, 1-dioxy thiomorpholinyl) imidazo [1,2-a ] pyridine-3-yl) phenyl) -5-nitrofuran-2-carboxamide (15) are as follows:

the preparation process comprises the following steps:

step 1: preparation of 4- (3- (4-nitrophenyl) imidazo [1,2-a ] pyridin-7-yl) thiomorpholine 1, 1-dioxide (15b)

The 1-methylpiperazine was replaced by 1, 1-dithiomorpholine, and the remaining required starting materials, reagents and preparation were the same as in example 13, step 1, giving a tan solid.¹H NMR(400MHz,Chloroform-d)δ8.29(d,J＝7.7Hz,1H),8.06– 7.96(m,2H),7.83–7.80(m,1H),7.37(dd,J＝6.8,4.7Hz,2H),6.75(dd,J＝7.9,2.5Hz,2H), 4.14(q,J＝7.1Hz,4H),3.74(d,J＝7.1Hz,4H)；MS(EI)m/z 373.08(M+H)⁺.

Step 2: preparation of 4- (3- (4-aminophenyl) imidazo [1,2-a ] pyridin-7-yl) thiomorpholine 1, 1-dioxide (15c)

The remaining required starting materials, reagents and preparation were the same as in example 1, step 3, replacing 1b with 15b, to give a tan solid.¹H NMR(400MHz,Chloroform-d)δ8.11(d,J＝7.7Hz,1H),7.44(s,1H),7.29(d,J＝ 2.0Hz,1H),7.27(s,1H),6.91(d,J＝2.4Hz,1H),6.83–6.77(m,2H),6.53(dd,J＝7.7,2.6Hz, 1H),3.93–3.87(m,4H),3.17–3.12(m,4H).¹³C NMR(126MHz,Chloroform-d)δ146.71, 146.64,144.25,131.15,129.38,125.11,124.52,118.80,115.48,105.57,99.51,50.31,47.18； MS(EI)m/z 343.63(M+H)⁺.

And step 3: preparation of N- (4- (7- (1, 1-dioxothiomorpholinyl) imidazo [1,2-a ] pyridin-3-yl) phenyl) -5-nitrofuran-2-carboxamide (15)

Changing 1c to 15c, and the rest of the required raw materials and reagentsAnd the preparation method is the same as the step 4 in the example 1, and a brownish yellow solid is obtained.¹H NMR(500MHz,DMSO-d₆)δ10.88(s,1H),8.42(d,J＝7.6Hz,1H),7.94(d,J＝8.4 Hz,2H),7.84(d,J＝3.9Hz,1H),7.76(d,J＝3.9Hz,1H),7.67–7.60(m,3H),6.99(s,2H),3.91(t, J＝5.0Hz,4H),3.21–3.17(m,4H).¹³C NMR(101MHz,DMSO-d₆)δ155.10,152.29,148.34, 147.49,145.66,137.68,131.34,130.11,127.91,125.28,123.75,121.71,117.22,113.95,106.65, 97.45,50.30,46.68；MS(EI)m/z 482.11(M+H)⁺；HRMS(ESI)calcd for C₂₂H₁₉N₅O₆S(M+H)⁺: 482.1129；found 482.1130.

Example 16

The structure and preparation process of N- (4- (7- (4-cyclopropylpiperazin-1-yl) imidazo [1,2-a ] pyridin-3-yl) phenyl) -5-nitrofuran-2-carboxamide (16) are as follows:

the preparation process comprises the following steps:

step 1: preparation of 7- (4-Cyclopropylpiperazin-1-yl) -3- (4-nitrophenyl) imidazo [1,2-a ] pyridine (16b)

The 1-methylpiperazine was replaced by 1-cyclopropylpiperazine, and the remaining required starting materials, reagents and preparation were the same as in example 13, step 1, giving a tan solid.¹H NMR(400MHz,DMSO-d₆)δ8.55(d,J＝7.7Hz,1H),8.30(d,J ＝8.7Hz,2H),7.91(d,J＝8.6Hz,2H),7.85(s,1H),7.03–6.96(m,1H),6.86(s,1H),3.26(d,J＝ 5.2Hz,4H),2.68(t,J＝5.0Hz,4H),2.13–1.90(m,1H),0.45(m,2H),0.36(m,2H)；MS(EI)m/z 364.16(M+H)⁺.

Step 2: preparation of 4- (7- (4-cyclopropylpiperazin-1-yl) imidazo [1,2-a ] pyridin-3-yl) aniline (16c)

The remaining required starting materials, reagents and preparation were the same as in example 1, step 3, replacing 1b with 16b, to give a tan solid.¹H NMR(500MHz,DMSO-d₆)δ8.34(d,J＝7.8Hz,1H),7.80(s,1H),7.28(d,J＝8.1 Hz,3H),6.97(s,1H),6.75(d,J＝8.2Hz,2H),2.04–1.95(m,1H),0.85(m,2H),0.62(s,2H)； MS(EI)m/z 334.19(M+H)⁺.

And step 3: preparation of N- (4- (7- (4-cyclopropylpiperazin-1-yl) imidazo [1,2-a ] pyridin-3-yl) phenyl) -5-nitrofuran-2-carboxamide (16)

The 1c was replaced with 16c and the remaining required starting materials, reagents and preparation were the same as in step 4 of example 1 to give a tan solid.¹H NMR(500MHz,DMSO-d₆)δ10.84(s,1H),8.39(d,J＝7.7Hz,1H),7.93(d,J＝8.3 Hz,2H),7.84(d,J＝3.9Hz,1H),7.71(d,J＝3.9Hz,1H),7.68–7.63(m,3H),6.99(dd,J＝7.8, 2.4Hz,1H),6.78(d,J＝2.4Hz,1H),3.28(t,J＝5.2Hz,4H),2.69(t,J＝5.0Hz,4H),1.71–1.66 (m,1H),0.49–0.44(m,2H),0.39–0.35(m,2H).¹³C NMR(126MHz,DMSO-d₆)δ155.12, 152.29,149.61,148.28,146.63,137.95,130.11,128.22,125.29,124.82,123.85,121.64,117.22, 113.97,107.25,94.60,52.76,47.67,38.45,6.10.；MS(EI)m/z 473.19(M+H)⁺；HRMS(ESI)calcd for C₂₅H₂₄N₆O₄(M+H)⁺:473.1932；found 473.1931.

Example 17

The structure and preparation process of N- (4- (7- (4-hydroxy-4-methylpiperidin-1-yl) imidazo [1,2-a ] pyridin-3-yl) phenyl) -5-nitrofuran-2-carboxamide (17) are shown in the following

The preparation process comprises the following steps:

step 1: preparation of 4-methyl-1- (3- (4-nitrophenyl) imidazo [1,2-a ] pyridin-7-yl) piperidin-4-ol (17b)

The 1-methylpiperazine was replaced by 4-methylpiperidin-4-ol and the remaining required starting materials, reagents and preparation were the same as in example 13, step 1 to give a tan solid.¹H NMR(500MHz,Chloroform-d)δ8.38–8.28(m,2H),8.23(d, J＝7.7Hz,1H),7.68-7.70(m,3H),6.90(d,J＝2.5Hz,1H),6.74(dd,J＝7.7,2.5Hz,1H),3.55– 3.47(m,2H),3.38–3.29(m,2H),1.77–1.73(m,4H),1.33(s,3H).¹³C NMR(126MHz, Chloroform-d)δ149.99,148.98,146.00,136.36,134.60,126.28,124.79,123.47,122.18,107.38, 97.63,67.65,44.77,37.80,30.20.MS(EI)m/z 353.15(M+H)⁺.

Step 2: preparation of 4-methyl-1- (3- (4-nitrophenyl) imidazo [1,2-a ] pyridin-7-yl) piperidin-4-ol (17c)

The material 1b was replaced with 17b and the remaining required starting materials, reagents and preparation were the same as in example 1, step 3, to give a tan solid.¹H NMR(500MHz,DMSO-d₆)δ8.13(d,J＝7.7Hz,1H),7.28(s,1H),7.21(d,J＝8.2 Hz,2H),6.81(dd,J＝7.8,2.5Hz,1H),6.70(m,3H),5.32(brs,2H),3.42–3.39(m,2H),3.22– 3.16(m,2H),1.60–1.54(m,4H),1.16(s,3H).¹³C NMR(126MHz,DMSO-d₆)δ148.87,147.92, 147.26,129.93,128.87,124.59,124.17,116.70,114.72,106.82,96.46,66.34,44.92,37.94,30.13. MS(EI)m/z 323.17(M+H)⁺.

And step 3: the structure for the preparation of N- (4- (7- (4-hydroxy-4-methylpiperidin-1-yl) imidazo [1,2-a ] pyridin-3-yl) phenyl) -5-nitrofuran-2-carboxamide (17) is shown below

The 1c was replaced with 17c and the remaining required starting materials, reagents and preparation were the same as in step 4 of example 1 to give a tan solid.¹H NMR(500MHz,DMSO-d₆)δ10.88(s,1H),8.33(d,J＝7.7Hz,1H),7.92(d,J＝8.2 Hz,2H),7.83(d,J＝3.8Hz,1H),7.75(d,J＝3.9Hz,1H),7.62(d,J＝8.2Hz,2H),7.51(s,1H), 6.87(d,J＝7.8Hz,1H),6.74(s,1H),3.44(d,J＝17.0Hz,2H),3.24–3.18(m,2H),1.57(t,J＝ 5.4Hz,4H),1.16(s,3H).¹³C NMR(126MHz,DMSO-d₆)δ155.05,152.27,148.37,148.30, 137.31,131.97,130.10,127.59,126.00,124.52,123.30,121.68,117.14,113.97,107.03,96.34, 66.35,44.75,37.89,30.14.MS(EI)m/z 462.17(M+H)⁺；HRMS(ESI)calcd for C₂₄H₂₃N₅O₅ (M+H)⁺:462.1772；found 462.1770.

Example 18

The structure and preparation process of N- (4- (7- (4-methyl-1, 4-diaza-1-yl) imidazo [1,2-a ] pyridin-3-yl) phenyl) -5-nitrofuran-2-carboxamide (18) are as follows:

the preparation process comprises the following steps:

step 1: preparation of 7- (4-methyl-1, 4-diaza-1-yl) -3- (4-nitrophenyl) imidazo [1,2-a ] pyridine (18b)

The 1-methylpiperazine was replaced by N-methylpiperazine, and the remaining required starting materials, reagents and preparation were the same as in example 13, step 1, giving a tan solid.

Step 2: 4- (7- (4-methyl-1, 4-diaza-1-yl) imidazo [1,2-a ] pyridin-3-yl) aniline (18c)

The procedure of example 1 was followed by replacing 1b with 18b and the remaining required starting materials, reagents and preparation were the same as in example 1, step 3, to give a tan solid.¹H NMR(500MHz,Chloroform-d)δ7.91(d,J＝7.7Hz,1H),7.24(s,1H),7.17(d,J＝ 8.2Hz,2H),6.69(d,J＝8.0Hz,2H),6.52(d,J＝2.6Hz,1H),6.36(dd,J＝7.8,2.6Hz,1H),4.20 –4.04(m,2H),3.53–3.49(m,2H),3.44(t,J＝6.3Hz,2H),2.64–2.60(m,2H),2.49–2.45(m, 2H),2.29(s,3H),1.95–1.90(m,2H).¹³C NMR(126MHz,Chloroform-d)δ146.97,145.41, 145.32,128.27,128.10,122.70,118.25,114.41,102.49,91.33,56.66,55.91,47.88,47.33,45.66, 26.42.MS(EI)m/z 322.19(M+H)⁺.

And step 3: preparation of N- (4- (7- (4-methyl-1, 4-diaza-1-yl) imidazo [1,2-a ] pyridin-3-yl) phenyl) -5-nitrofuran-2-carboxamide (18)

The procedure of example 1 was repeated except for changing 1c to 18c and the remaining required starting materials, reagents and preparation were the same as in step 4 to give a brown-yellow solid.¹H NMR(400MHz,DMSO-d₆)δ11.25(s,1H),8.33(d,J＝7.7Hz,1H),8.11(d,J＝3.9 Hz,1H),8.02(d,J＝8.3Hz,2H),7.83(d,J＝3.9Hz,1H),7.59(d,J＝8.3Hz,2H),7.47(s,1H), 6.75(d,J＝7.8Hz,1H),6.53(s,1H),3.53–3.48(m,4H),2.70(t,J＝4.9Hz,2H),2.55(d,J＝5.0 Hz,2H),2.31(s,3H),1.96–1.91(m,2H)；MS(EI)m/z 461.19(M+H)⁺；HRMS(ESI)calcd for C₁₉H₁₃N₄O₅F(M+H)⁺:461.1932；found 461.1933.

Example 19

The structure and preparation process of N- (4- (7- (4-hydroxy-4-methylpiperidin-1-yl) imidazo [1,2-a ] pyridin-3-yl) phenyl) -5-nitrofuran-2-carboxamide (19) are as follows:

the preparation process comprises the following steps:

step 1: preparation of 3- (4-Nitrobenzene) -7- (4- (oxyethan-3-yl) piperazin-1-yl) imidazo [1,2-a ] pyridine (19b)

The 1-methylpiperazine was replaced by 1- (3-oxetanyl) piperazine and the remaining required starting materials, reagents and preparation were the same as in example 13, step 1, giving a tan solid.¹H NMR(400MHz,Chloroform-d)δ8.35(d,J＝8.3Hz,2H), 8.25(d,J＝7.6Hz,1H),7.69(m,3H),7.27(s,2H),6.91(s,1H),6.74(d,J＝7.7Hz,1H),4.69(dt, J＝23.6,6.3Hz,4H),3.57(m,1H),3.37(t,J＝4.9Hz,4H),2.53(t,J＝4.9Hz,4H).MS(EI)m/z 380.16(M+H)⁺.

Step 2: preparation of 4- (7- (4- (oxyethan-3-yl) piperazin-1-yl) imidazo [1,2-a ] pyridin-3-yl) aniline (19c)

The procedure of example 1 was followed by changing 1b to 19b and the remaining required starting materials, reagents and preparation were the same as in example 1, step 3, to give a tan solid.¹H NMR(400MHz,DMSO-d₆)δ8.31(d,J＝7.9Hz,1H),7.78(s,1H),7.27(d,J＝8.4 Hz,3H),6.94(d,J＝2.5Hz,1H),6.78–6.71(m,2H),4.59(q,J＝6.3Hz,4H),3.57(s,4H),3.39 (s,1H),2.57(s,4H).MS(EI)m/z 350.19(M+H)⁺.

And step 3: preparation of N- (4- (7- (4-hydroxy-4-methylpiperidin-1-yl) imidazo [1,2-a ] pyridin-3-yl) phenyl) -5-nitrofuran-2-carboxamide (19)

The procedure of example 1 was followed by changing 1c to 19c and the remaining required starting materials, reagents and preparation to give a tan solid.¹H NMR(400MHz,DMSO-d₆)δ10.86(s,1H),8.42(d,J＝7.8Hz,1H),7.95(d,J＝8.4 Hz,2H),7.84(d,J＝3.9Hz,1H),7.76(s,1H),7.71(d,J＝4.0Hz,1H),7.67(d,J＝8.6Hz,2H), 7.08(dd,J＝7.9,2.4Hz,1H),6.83(s,1H),4.59(t,J＝6.5Hz,2H),4.49(t,J＝6.0Hz,2H),3.41 (m,5H),2.43(t,J＝5.1Hz,4H).¹³C NMR(101MHz,DMSO)δ155.22,152.32,151.01,148.19, 138.88,130.33,130.16,128.97,126.23,124.47,123.28,122.14,117.35,113.97,107.67,94.04, 74.73,58.77,49.02,46.94.MS(EI)m/z 489.18(M+H)⁺；HRMS(ESI)calcd for C₂₅H₂₄N₆O₅ (M+H)⁺:489.1881；found 489.1885.

Example 20

The structure and preparation process of tert-butyl 4- (3- (4- (5-nitrofuran-2-carboxamide) phenyl) imidazo [1,2-a ] pyridin-7-yl) piperazine-1-carboxylate (20) are as follows:

the preparation process comprises the following steps:

step 1: preparation of tert-butyl 4- (3- (4-nitrophenyl) imidazo [1,2-a ] pyridin-7-yl) piperazine-1-carboxylate (20b)

The 1-methylpiperazine was replaced by piperazine-1-carboxylic acid tert-butyl ester, and the remaining required raw materials, reagents and preparation method were the same as in example 13, step 1, yielding a tan solid.¹H NMR(400MHz,Chloroform-d)δ8.36(d,J＝8.8Hz,2H),8.27 (d,J＝7.7Hz,1H),7.75–7.67(m,3H),6.92(d,J＝2.4Hz,1H),6.77–6.70(m,1H),3.64(t,J＝ 5.2Hz,4H),3.29(t,J＝5.2Hz,4H),1.52(s,9H).¹³C NMR(126MHz,Chloroform-d)δ154.57, 149.44,148.95,146.22,136.14,134.57,126.55,124.80,123.62,122.44,107.39,98.43,80.33, 48.14,43.22,27.67.MS(EI)m/z 424.19(M+H)⁺.

Step 2: 4- (3- (4-aminophenyl) imidazo [1,2-a ] pyridin-7-yl) piperazine-1-carboxylic acid tert-butyl ester (20c)

The remaining required starting materials, reagents and preparation were the same as in example 1, step 3, replacing 1b with 20b, to give a tan solid.¹H NMR(400MHz,Methanol-d₄)δ8.01(d,J＝7.5Hz,1H),7.25(s,1H),7.16(d,J＝8.1 Hz,2H),6.79(d,J＝8.1Hz,2H),6.69–6.62(m,2H),3.49(t,J＝5.3Hz,4H),3.14(d,J＝5.3Hz, 4H),1.47(s,9H).¹³C NMR(101MHz,Methanol-d₄)δ154.82,149.01,148.35,146.57,128.83, 127.24,125.36,124.00,116.79,115.11,106.59,95.14,80.09,43.49,42.41,27.36.MS(EI)m/z 394.21(M+H)⁺.

And step 3: preparation of tert-butyl 4- (3- (4- (5-nitrofuran-2-carboxamide) phenyl) imidazo [1,2-a ] pyridin-7-yl) piperazine-1-carboxylate (20)

The 1c was replaced with 20c and the remaining required starting materials, reagents and preparation were the same as in step 4 of example 1 to give a tan solid.¹H NMR(400MHz,DMSO-d₆)δ10.90(s,1H),8.50(d,J＝7.7Hz,1H),7.99(d,J＝8.1 Hz,3H),7.85(d,J＝3.9Hz,1H),7.71(dd,J＝6.3,2.3Hz,3H),7.22(d,J＝7.9Hz,1H),6.86(s, 1H),3.57–3.49(m,8H),1.44(s,9H).¹³C NMR(126MHz,DMSO-d₆)δ155.26,154.29,152.32, 152.03,148.12,143.05,139.37,129.56,127.16,122.16,121.57,117.39,113.96,108.00,89.23, 79.76,54.06,46.43,28.50.MS(EI)m/z 533.20(M+H)⁺；HRMS(ESI)calcd for C₂₇H₂₉N₆O₆ (M+H)⁺:533.2068；found 533.2064.

Example 21

The structure and preparation process of 5-nitro-N- (4- (7- (piperazine-1-yl) imidazo [1,2-a ] pyridin-3-yl) phenyl) furan-2-carboxamide (21) are as follows:

the preparation process comprises the following steps:

step 1: preparation of 5-nitro-N- (4- (7- (piperazin-1-yl) imidazo [1,2-a ] pyridin-3-yl) phenyl) furan-2-carboxamide (21)

Compound 20(50mg, 0.09mmol) was dissolved in 5mL of dichloromethane, and 1mL of trifluoroacetic acid was added dropwise to the above reaction solution at room temperature. After stirring at room temperature for 1 hour, the solution was diluted with 50mL of dichloromethane. Saturated sodium bicarbonate solution was added under ice bath and adjusted to pH 7. The organic layer was washed with brine and anhydrous sulfuric acidAnd (4) drying sodium. The resulting solution was evaporated and purified by silica gel column chromatography (dichloromethane/methanol 50:3, V/V) to give the desired product (30mg, 77%) as a yellow-brown solid.¹H NMR(500MHz,DMSO-d₆)δ10.89(s,1H),8.50(d,J＝7.8Hz,1H),7.99(t,J ＝4.5Hz,3H),7.85(d,J＝3.9Hz,1H),7.70(dd,J＝10.0,6.1Hz,3H),7.23(d,J＝8.2Hz,1H), 6.86(s,1H),3.54(t,J＝10.1Hz,8H).MS(EI)m/z 433.15(M+H)⁺；HRMS(ESI)calcd for C₂₀H₂₁N₆O₄(M+H)⁺:433.1540；found 433.1544.

Example 22

The structure and preparation process of tert-butyl 4- (3- (4- (5-nitrofuran-2-carboxamide) phenyl) imidazo [1,2-a ] pyridin-7-yl) -3, 6-dihydropyridine-1 (2H) -carboxylate (2) are as follows:

the preparation process comprises the following steps:

step 1: preparation of 4- (3- (4-Nitrophenyl) imidazo [1,2-a ] pyridin-7-yl) -3, 6-dihydropyridine-1 (2H) -carboxylic acid tert-butyl ester (22b)

Compound 11b (736mg, 2mmol) and N-Boc-1,2,5, 6-tetrahydropyridine-4-boronic acid pinacol ester (679.8mg, 2.2mmol) were dissolved in 10mL of a 1, 4-dioxane/water (3:1, V/V) solution, and potassium carbonate (424mg, 4mmol) and [1,1' -bis (diphenylphosphino) ferrocene were dissolved at room temperature]Palladium dichloride dichloromethane complex (81.6mg, 0.1mmol) was added to the above reaction solution. After stirring at 110 ℃ for 12h, the reaction was diluted with 100ml of ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. The resulting solution was evaporated and purified by silica gel column chromatography (dichloromethane/methanol 50:1, V/V) to give 545mg of a brown solid, yield, 65%.¹H NMR(500MHz,Chloroform-d)δ8.12(d,J＝7.4Hz,1H), 7.30(d,J＝9.0Hz,2H),7.26(d,J＝8.0Hz,2H),6.90(d,J＝7.4Hz,1H),6.81(d,J＝7.9Hz,2H), 6.21(s,1H),4.11(s,2H),3.56(d,J＝6.0Hz,2H),2.44(s,2H),1.49(s,9H).MS(EI)m/z 421.18 (M+H)⁺.

Step 2: 4- (3- (4-Aminophenyl) imidazo [1,2-a ] pyridin-7-yl) -3, 6-dihydropyridine-1 (2H) -carboxylic acid tert-butyl ester (22c)

The remaining required starting materials, reagents and preparation were the same as in example 1, step 3, replacing 1b with 22b, to give a tan solid.¹H NMR(500MHz,Chloroform-d)δ8.16(d,J＝7.4Hz,1H),7.55(d,J＝9.0Hz,2H), 7.31(d,J＝8.0Hz,2H),6.90(d,J＝7.4Hz,1H),6.81(d,J＝7.9Hz,2H),6.21(s,1H),4.13(s, 2H),3.66(d,J＝6.0Hz,2H),2.56(s,2H),1.50(s,9H).MS(EI)m/z 391.21(M+H)⁺.

And step 3: preparation of tert-butyl 4- (3- (4- (5-nitrofuran-2-carboxamide) phenyl) imidazo [1,2-a ] pyridin-7-yl) -3, 6-dihydropyridine-1 (2H) -carboxylate (2)

The 1c was replaced with 22c and the remaining required starting materials, reagents and preparation were the same as in step 4 of example 1 to give a tan solid.¹H NMR(400MHz,Chloroform-d)δ8.48(s,1H),8.25(d,J＝7.3Hz,1H),7.79(d,J＝ 8.2Hz,2H),7.67(s,1H),7.63(s,1H),7.52(s,1H),7.47(d,J＝8.1Hz,2H),7.24(d,J＝3.2Hz, 1H),7.14(d,J＝7.1Hz,1H),6.54(s,1H),6.31(s,1H),4.11(s,2H),3.62(s,2H),2.50(s,2H), 1.48(s,9H).MS(EI)m/z 530.19(M+H)⁺；HRMS(ESI)calcd for C₂₈H₂₈N₅O₆(M+H)⁺:530.1968； found 530.1964.

Example 23

The structure of 5-nitro-N- (4- (7- (1,2,3, 6-tetrahydropyridin-4-yl) imidazo [1,2-a ] pyridin-3-yl) phenyl) furan-2-carboxamide (23) is shown below:

the preparation process comprises the following steps:

step 1: preparation of 5-nitro-N- (4- (7- (1,2,3, 6-tetrahydropyridin-4-yl) imidazo [1,2-a ] pyridin-3-yl) phenyl) furan-2-carboxamide (23)

The 20 was replaced with 22 and the remaining required starting materials, reagents and preparation were the same as in example 21, step 1, giving a tan solid.¹H NMR(400MHz,DMSO-d₆)δ11.22(s,1H),9.62(s,1H),8.56(d,J＝7.4Hz,1H), 8.06(m,3H),7.85(d,J＝3.9Hz,1H),7.80(s,1H),7.70(d,J＝8.5Hz,2H),7.67(s,1H),7.21(d,J ＝7.0Hz,1H),6.48(s,1H),3.83–3.72(m,2H),3.05–2.59(m,2H),2.51(s,2H).¹³C NMR(126 MHz,DMSO-d₆)δ155.13,152.29,148.30,146.47,137.93,136.52,133.39,132.60,128.20,125.43, 125.37,125.21,123.99,121.68,117.21,113.97,111.85,110.48,45.01,42.59,25.99.MS(EI)m/z 430.14(M+H)⁺；HRMS(ESI)calcd for C₂₃H₂₀N₅O₄(M+H)⁺:430.1437；found 430.1435.

Example 24

The structure and preparation process of N- (4- (7- (1-acetyl-1, 2,3, 6-tetrahydropyridin-4-yl) imidazo [1,2-a ] pyridin-3-yl) phenyl) -5-nitrofuran-2-carboxamide (24) are as follows:

the preparation process comprises the following steps:

step 1: preparation of N- (4- (7- (1-acetyl-1, 2,3, 6-tetrahydropyridin-4-yl) imidazo [1,2-a ] pyridin-3-yl) phenyl) -5-nitrofuran-2-carboxamide (24)

The remaining required starting materials, reagents and preparation were the same as in example 1, step 4, replacing 1c with 30, to give a tan solid.¹H NMR(400MHz,DMSO-d₆)δ8.53(d,J＝7.4Hz,1H),8.16(m,3H),7.85(d,J＝3.9Hz, 1H),7.81(s,1H),7.70(d,J＝8.5Hz,2H),7.64(s,1H),7.18(d,J＝7.0Hz,1H),6.48(s,1H),3.83 –3.72(m,2H),3.05–2.59(m,2H),2.51(s,2H),2.31(s,3H).MS(EI)m/z 472.15(M+H)⁺； HRMS(ESI)calcd for C₂₅H₂₂N₅O₅(M+H)⁺:472.1544；found 472.1539.

Example 25

The structure and preparation process of N- (4- (7- (1-methyl-1H-pyrazol-4-yl) imidazo [1,2-a ] pyridin-3-yl) phenyl) -5-nitrofuran-2-carboxamide (25) are shown as follows:

the preparation process comprises

Step 1: preparation of 7- (1-methyl-1H-pyrazol-4-yl) -3- (4-nitrophenyl) imidazo [1,2-a ] pyridine (25b)

The N-Boc-1,2,5, 6-tetrahydropyridine-4-boronic acid pinacol ester was replaced with 1-methyl-4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxobenzaldehyde-2-yl) -1H-pyrazole, and the remaining required starting materials, reagents and preparation were the same as in step 1 of example 22 to give a tan solid.¹H NMR(500MHz,DMSO-d₆)δ8.46(s,1H),8.07(s,1H),8.19(d,J＝8.2Hz,2H),7.99 (m,2H),7.71(s,1H),7.68(d,J＝8.5Hz,2H),7.21(d,J＝7.2Hz,1H),3.91(s,3H)；MS(EI)m/z 320.10(M+H)⁺；

Step 2: preparation of 4- (7- (1-methyl-1H-pyrazolyl-4-yl) imidazo [1,2-a ] pyridin-3-yl) aniline (25c)

The remaining required starting materials, reagents and preparation were the same as in example 1, step 3, replacing 1b with 25b, to give a tan solid.¹H NMR(500MHz,DMSO-d₆)δ8.36(s,1H),8.05(s,1H),8.01(d,J＝8.2Hz,2H),7.86 (m,2H),7.71(s,1H),7.69(d,J＝8.5Hz,2H),7.20(d,J＝7.2Hz,1H),3.90(s,3H)；MS(EI)m/z 290.13(M+H)⁺；

And step 3: preparation of N- (4- (7- (1-methyl-1H-pyrazol-4-yl) imidazo [1,2-a ] pyridin-3-yl) phenyl) -5-nitrofuran-2-carboxamide (25)

The 1c was replaced with 25c and the remaining required starting materials, reagents and preparation were the same as in step 4 of example 1 to give a tan solid.¹H NMR(500MHz,DMSO-d₆)δ11.12(s,1H),8.56(d,J＝7.2Hz,1H),8.36(s,1H), 8.08(s,1H),8.03(d,J＝8.2Hz,2H),7.97(d,J＝2.9Hz,1H),7.89–7.84(m,2H),7.73(s,1H), 7.70(d,J＝8.5Hz,2H),7.22(d,J＝7.2Hz,1H),3.90(s,3H)；MS(EI)m/z 429.12(M+H)⁺； HRMS(ESI)calcd for C₂₂H₁₇N₆O₄(M+H)⁺:429.1230；found 429.1230.

Example 26

The structure and preparation process of N- (4- (imidazo [1,2-a ] pyridin-3-yl) phenyl) -5-nitrofuran-2-carboxamide (26) are shown as follows:

the preparation process comprises the following steps:

step 1: the structure for the preparation of N- (4- (imidazo [1,2-a ] pyridin-3-yl) phenyl) -5-nitrofuran-2-carboxamide (26c) is shown below

By replacing 1c with 4- (imidazo [1,2-a ]]Pyridin-3-yl) aniline, the remaining required starting materials, reagents and preparation were the same as in example 1, step 4, to give a tan solid.¹H NMR(400MHz,DMSO-d₆)δ10.82(s,1H),8.59(d,J＝ 6.9Hz,1H),7.95(d,J＝8.6Hz,2H),7.83(d,J＝3.9Hz,1H),7.78(s,1H),7.71(s,1H),7.70– 7.68(m,2H),7.66(d,J＝9.1Hz,1H),7.31(q,1H),6.98(t,J＝6.5Hz,1H).MS(EI)m/z 349.08 (M+H)⁺；HRMS(ESI)calcd for C₁₈H₁₃N₄O₄(M+H)⁺:349.0854；found 349.0856.

Biological Activity test section

It was found that compounds 1-19 and compound 23 were able to inhibit the proliferation of a variety of cancer cells, including breast, lung and gastric cancer cells.

The compound 23 small molecule compound can effectively inhibit the tyrosine phosphorylation level of STAT3, and inhibit the transcriptional activity of STAT3 and the expression of downstream target genes Bcl-xl, C-myc and Mcl-1 thereof.

The compound 23 small molecular compound can obviously inhibit the growth of gastric cancer in a mouse subcutaneous cell transplantation tumor model, reduces the tumor body weight and has no obvious toxicity to mouse organs.

Application example 1: compounds 1-19 and 23 inhibit breast, lung and gastric cancer cell proliferation

(1) Cell culture

MDA-MB-231 and MCF-7 cells are cultured in DMEM medium containing 10% fetal calf serum and 1% double-resistant penicillin and streptomycin, lung cancer cells A549 and PC9, gastric cancer cells AGS and MGC803. Melanoma cells MP41 and 92-1, leukemia cells MOLM-13 and MV4-11, and prostate cancer cell C42, culturing in RPIM-1640 medium containing 10% fetal calf serum and 1% double-resistant penicillin and streptomycin, standing at 37 deg.C and 5% CO₂In a cell culture incubator.

(2) CCK8 method for determining cell viability

1) Taking each cell in logarithmic phase, digesting the cell, counting, uniformly inoculating the cell in a 96-well plate at the density of 100 mu L per well of (1-3) x 10000/well, and placing the cell in a constant temperature incubator until the cell adheres to the wall;

2) after adherence, adding corresponding culture medium into a control group, only adding the culture medium into a blank group without cells, and adding various compounds with different concentrations into an administration group;

3) after the drug treatment is carried out for 72h, the cell state is observed under a microscope, CCK8(10 mu L/hole) is added under the condition of keeping out of the sun, and the mixture is evenly mixed and placed in an incubator at 37 ℃ in the absence of the light for incubation for 0.5-4 h;

4) after air bubbles are removed, reading the light absorption value (OD) at the position of 450nm by using enzyme labeling, and repeating the experiment for three times;

5) cell viability (%) ═ a (compound) -a (blank) ]/[ a (control) -a (blank) ] × 100%;

6) finally, calculation was performed using GraphPad Prism8 software to obtain IC 50.

(3) Results of the experiment

The experimental results are shown in table 1, and the compounds 1 to 19 and the compound 23 have good activity on 4 cancer cells and show good inhibition effect; FIG. 1 shows the growth inhibition curves and IC50 of compound 23 on various cancer cells, including lung cancer cells A549 and PC9, gastric cancer cells AGS and MGC803, melanoma cells MP41 and 92-1, leukemia cells MOLM-13 and MV4-11, and prostate cancer cell C42.

TABLE 1 inhibitory Activity of Compounds 1-19 and 23 against 4 cancer cells

Application example 2: compound 23 is capable of inhibiting phosphorylation of STAT3 and dimer formation

The present application example uses compound 23 as an example, and measures the effect of inhibiting phosphorylation of STAT3 and formation of a dimer. The specific process is as follows:

(1) western blotting (Western Blot)

After the cells are treated by the compound 23 with different concentrations for 24 hours, the culture medium is sucked off, the cell lysate RIPA containing protease inhibitor and phosphatase inhibitor is added to lyse the cells, then the lysate is centrifuged for 15min at 15000rpm and 4 ℃, the supernatant is taken out for quantification, and 5 × loading buffer is added to the lysate for boiling denaturation. Then separating protein samples by using polyacrylamide gel SDS-PAGE electrophoresis, transferring the protein samples to a nitrocellulose membrane (PVDF membrane), after blocking for 1h by 5% BSA, respectively incubating overnight at 4 ℃ by using primary antibodies of pY705-STAT3, STAT3, C-myc, Bcl-xl, Mcl1 and beta-Actin, respectively, then incubating for 1h at room temperature by using rabbit secondary antibodies and mouse secondary antibodies with fluorescent markers, and finally detecting the expression level of the protein by using a Tanon developing instrument.

(2) Results of the experiment

Experimental results as shown in fig. 2, fig. (2A) shows that compound 23 is capable of time-dependently inhibiting phosphorylation of STAT3(pY 705); figure (2B) shows that compound 23 is able to inhibit STAT3(pY705) phosphorylation concentration-dependently. Application example 3: compound 23 can inhibit STAT3 transcriptional activity and target gene expression

In this application, compound 23 was used as an example, and the transcriptional activity of STAT3 and the expression effect of the target gene were measured. The specific process is as follows:

(1) western blotting method is as above

(2) Dual luciferase reporter gene assay

1) Taking 293T cells in a logarithmic growth phase, digesting the cells, counting the cells, and inoculating the cells into a 96-well plate at the density of 1-2 x 10000 cells/100 mu L;

2) culturing the cells in an incubator at 37 ℃ overnight, and performing a cell transfection experiment when the cell fusion degree is about 80%;

3) a, B two mixed solutions were prepared in 1.5mL centrifuge tubes (hereinafter, the amount of each well)

A: 0.25. mu.L Lipo 2000+ 5. mu.L OPTI-MEMI Medium

B: 50ng pGL3-STAT3-promoter plasmid +50ng STAT3C plasmid +40ng TKRL (Renilla luciferin reporter plasmid) + 5. mu.L OPTI-MEM Medium

Mixing solution A and solution B, and standing at room temperature for 5 min;

4) gently mixing the A, B solutions, and standing at room temperature for 15 min;

5) adding the mixed solution into each hole of a 96-hole plate along the 45-degree angle of the wall;

6) putting 96-well plate cells into a cell culture box, incubating for 24h, and adding compounds with different concentrations for 23 treatment for 24 h;

7) dissolving firefly luciferase detection reagent and Renilla luciferase detection buffer solution to room temperature, and placing Renilla luciferase detection substrate (100X) on an ice bath for later use;

8) taking a proper amount of renilla luciferase detection buffer solution according to the dosage of 100 mu Lyongli per hole, and adding a renilla luciferase detection substrate (100X) according to the ratio of 1:100 to prepare renilla luciferase detection working solution;

9) taking out the 96-well plate, discarding the culture medium, adding 50 μ L of reporter gene cell lysate into each well, shaking and mixing for 5 min;

10) adding 50 mu L of firefly luciferase detection reagent into each hole, and shaking and uniformly mixing for 5 min;

11) detecting to obtain RLU 1;

12) after the step of measuring the firefly luciferase is completed, 100 mu L of renilla luciferase detection working solution is added into each hole, and the solution is shaken and uniformly mixed for 5 min;

13) detecting to obtain RLU 2;

14) the ratio RLU1/RLU2 was obtained.

(3) Results of the experiment

The experimental results are shown in fig. 3, and fig. 3A shows that compound 23 can significantly inhibit the transcriptional activity of STAT 3; FIG. 3B shows that Compound 23 is capable of downregulating phosphorylation of STAT3(pY705) and its protein expression of the target genes C-myc, Bcl-xl, and Mcl-1.

Application example 4: the compound 23 can obviously inhibit the growth of tumors in stomach cancer bodies

In this application, compound 23 was used as an example to measure inhibition of tumor growth in stomach cancer. The specific process is as follows:

(1) subcutaneous tumor-bearing experiment in nude mice

1) The gastric cancer cells MGC803 in the logarithmic growth phase are taken, digested and counted, precooled PBS and Matrigel are mixed according to the proportion of 1:1, and the cells are resuspended to obtain 2 x 106 cells/100 mu L of cell suspension which is placed on ice. Injecting 100 mu L of cell suspension into subcutaneous parts on two sides of the abdomen and the back of a 4-5-week-old immunodeficient nude mouse;

2) when the subcutaneous tumor volume was about 100mm3, the subcutaneous tumors were randomly divided into three groups. The control group was administered with drug solvent (20% castor oil in PBS), the low dose group was administered with 3mg/kg of compound 23, and the high dose group was administered with 10mg/kg of compound 23. The administration mode is intraperitoneal injection, the administration volume is 100 mu L/unit, the administration is carried out once a day, and the administration is carried out continuously for four weeks. The mouse body weight and tumor size were measured every two days and mouse body weight gain and tumor growth curves were plotted.

3) Four weeks after dosing, mice were sacrificed for dissection, subcutaneous tumors were dissected, weighed and photographed.

(2) Results of the experiment

The results of the experiment are shown in fig. 4, where (4A) is the final volume size of representative tumors in each group after four weeks of administration; fig. 4B is a graph of tumor volume change during dosing, demonstrating that compound 23 is able to significantly inhibit the growth of gastric cancer tumors dose-dependently; FIG. 4C shows that Compound 23 is capable of dose-dependently reducing gastric cancer tumor weight, with a tumor inhibition of about 37.2% in the 3mg/kg group and about 78.86% in the 10mg/kg group; FIG. 4D is a graph showing the body weight change of nude mice during administration, indicating that Compound 23 has no effect on the body weight of nude mice.

Application example 5: compound 23 inhibits expression of pY705-STAT3 protein and target gene in vivo

In this application, the compound 23 was used as an example, and the inhibition of the expression of pY705-STAT3 protein and the target gene in vivo was measured. The specific process is as follows:

(1) hematoxylin-eosin staining method (HE)

The heart, liver, spleen, lung, kidney and subcutaneous tumors of each group of mice were stripped, washed with PBS, fixed with paraformaldehyde, dehydrated, paraffin-embedded, sliced, stained with hematoxylin and eosin, dehydrated and mounted, observed under a microscope and photographed, and the effect of compound 23 on visceral tissues and gastric cancer tumors was examined.

(2) Western blotting experiment (Western Blot)

Tumor tissues (about 20mg) of mice cryopreserved with liquid nitrogen were removed, thawed on ice, and 200. mu.L of RIPA lysate (containing protease inhibitor and phosphatase inhibitor) was added to cut the tissue pieces into small pieces with tissue scissors on ice. And (3) fully cracking the tissue blocks by using ice ultrasound (Apml 45%), stopping ultrasound when no tissue blocks are visible to the naked eyes, centrifuging at the temperature of 4 ℃ and the speed of 15000rpm for 15-20min, taking supernatant into a new centrifuge tube, quantifying BCA, and carrying out subsequent experiments by using the Western blot as described above.

(3) Results of the experiment

Experimental results as shown in fig. 5, fig. (5A) shows that compound 23 is also able to inhibit phosphorylation of STAT3(pY705) and protein expression of its target gene in mouse in vivo experiments; FIG. 5B is a HE diagram showing the tumor exfoliated in groups of nude mice, showing significant increase in tumor tissue space and cell arrangement disorder in the group to which compound 23 was administered; FIG. 5C is a HE chart of heart, liver, spleen, lung and kidney peeled from each group of nude mice, showing that the cell arrangement distribution, shape and the like of each tissue are not significantly different among the groups.

It should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the scope of the present invention, and that those skilled in the art can make other variations or modifications based on the above description and ideas, and all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.